CN114380813B

CN114380813B - VISTA targeted probe and preparation method and application thereof

Info

Publication number: CN114380813B
Application number: CN202210051416.0A
Authority: CN
Inventors: 郭志德; 刘佳; 张现忠; 文雪君; 方建阳
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2022-01-17
Filing date: 2022-01-17
Publication date: 2023-06-06
Anticipated expiration: 2042-01-17
Also published as: CN114380813A

Abstract

A VISTA targeted probe, a preparation method and application thereof relate to the field of medicinal chemistry, and a targeted compound is formed by connecting a VISTA inhibitor molecule and a nuclide labeling group in a specific chemical form. The invention also provides a radionuclide-labeled diagnosis and treatment probe based on the targeting compound, which is prepared by a wet method or a freeze-drying method. The invention also relates to the application of the probe as a diagnosis and treatment reagent in human or animal tumor or immune diseases, has the advantages of simple preparation, good stability, high focus uptake and the like, and is suitable for clinical popularization.

Description

VISTA targeted probe and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a compound of a T cell activation inhibitor (VISTA) of a targeted V-type immunoglobulin structural domain and a nuclide labeled disease diagnosis and treatment probe thereof.

Background

The development of immunology brings revolutionary changes to the treatment of major diseases such as tumors, and has become another effective treatment means for cancers after surgery, radiotherapy, chemotherapy and targeted treatment. Immunotherapeutic strategies represented by PD-1/PD-L1 immunosuppression pathway block and CAR-T cell therapy have brought benefit to oncology patients. But limited by the heterogeneity of the immune microenvironment, the therapeutic effect is not yet maximized. The T cell activation inhibitor of the type V immunoglobulin domain (VISTA, V-Domain Immunoglobulin-Containing Suppressor of T Cell Activation) is a very attractive target. VISTA, also known as PD-1H (Programmed de-1 homolog), belongs to the immunoglobulin family, and the extracellular domain is homologous to PD-L1, an immunomodulatory receptor expressed on most cell subsets of the hematopoietic lineage, and is often considered an indicator of poor prognosis for a variety of cancers. VISTA is a novel immunoregulatory molecule that down-regulates T cell responses. The broad expression of VISTA means that the VISTA has important steady-state effects in terms of regulating immune system reaction and distinguishing the prospects of immunoregulatory receptors, has higher non-redundant activity compared with other immune checkpoints, is a tumor immunotherapy target with great prospect, and is more and more important in terms of regulating T cell activity and tumor immunity. In addition to the nature of the tumor microenvironment, one promising aspect of VISTA targeted therapy is that the inhibitory activity of VISTA is independent of the PD-1/PD-L1 pathway. In recent years, immune checkpoints, such as PD-1 or CTLA4, play an important role in controlling T cell tolerance. However, they often begin expression after the T cells are activated. The mechanism by which immune checkpoints play a role in maintaining quiescence and tolerance of naive T cells is ambiguous. VISTA, a novel immune checkpoint, begins to persist in naive T cells. Therefore, knocking out the VISTA gene can greatly up-regulate the anti-tumor immunity mediated by T cells, and similarly, the blocking of the VISTA and the PD-1/PD-L1 can enhance the tumor immunity function.

In addition, VISTA can also play an important role as a target of immune response in autoimmune diseases, in addition to cancer, and the target is characterized in that cancer can be treated with blocking antibodies at the same time, and autoimmune diseases can be treated with agonist antibodies. In the future, knowledge of which autoimmune diseases highly express VISTA when cancer is treated by blocking VISTA may help to predict possible organ-specific immune related adverse events. In addition, an increasingly clear understanding of VISTA ligands or receptors would be advantageous for subsequent drug development. Although the important role played by VISTA has attracted strong attention, most related studies are still based on pathological tissue samples, and lack of visual assessment means and specific probes at the living body level greatly hinders the research progress of the target. In addition, the daunting complexity of immunobiology contradicts the need to provide patients with the most effective immunotherapy as soon as possible. Along with the continuous and deep development of personalized treatment and accurate immune research, the development of a novel radioactive diagnosis and treatment probe aiming at an immune key target VISTA is significant. In vivo imaging diagnosis is a major issue in diagnostic research, relative to in vitro diagnosis, due to its non-invasive, high sensitivity, low toxicity and rapid diagnostic capabilities. Imaging means represented by Immuno-PET/SPECT provides a sharp instrument for timely and accurately evaluating the change of microenvironment in the process of occurrence, development and evolution of diseases and formulating a more accurate immunotherapy scheme. By utilizing a high-resolution molecular imaging technology and combining a high-sensitivity high-specificity molecular probe, the tumor lesion condition is hopefully reflected from the molecular level, and the method has the advantages which cannot be achieved by biopsy and biomarker detection. In particular, in recent years, most immune checkpoint inhibitors and their corresponding radionuclide-labeled probes are monoclonal antibody-based drugs, and their long blood half-life becomes a limiting factor for further application. At present, the traditional Chinese medicine composition is widely applied clinically ¹⁸ F-labeled fluorodeoxyglucose ¹⁸ F-FDG) -PET imaging has become an important tool for tumor diagnosis, staging and efficacy monitoring. Unfortunately, the process of the present invention, ¹⁸ high F-FDG in normal heart brain tissueIngestion, inability to distinguish immune targets, susceptibility to low expression levels of glucose transporter in patients, etc. become a significant disadvantage of this imaging technique. Therefore, the development of new radionuclide probes for VISTA targets is necessary. In view of the remarkable therapeutic effect of the VISTA targeted drug in various preclinical tumor models, the VISTA visualization on the living body level is realized through the more flexible and changeable small molecule immunity detection point inhibitor probes, and the defects and blank existing in the target research are solved.

Disclosure of Invention

The invention aims to solve the problems in the prior art and provides a VISTA targeted probe, and a preparation method and application thereof.

In order to achieve the above purpose, the invention adopts the following technical scheme:

a VISTA-targeted probe is based on a VISTA compound having the structure shown in formula (1):

wherein R is ₁ For labeling groups with nuclides, the nuclides include ¹⁷⁷ Lu、 ⁹⁰ Y、 ¹⁸ F、 ⁶⁴ Cu、 ⁶⁸ Ga、 ⁶² Cu、 ⁶⁷ Cu、 ⁶⁴ Gd、 ⁸⁶ Y、 ⁸⁹ Zr、 ^99m Tc、 ⁸⁹ Sr， ¹⁵³ Sm、 ¹⁴⁹ Tb、 ¹⁶¹ Tb、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ²¹² Pb、 ²¹³ Bi、 ²²³ Ra、 ²²⁵ Ac、 ²²⁶ Th、 ²²⁷ Th、 ^{123/124/125/131} I、 ²¹¹ At or ¹¹¹ At least one of In.

The nuclide is selected from ⁶⁸ Ga、 ^99m Tc、 ¹⁸ F、 ¹⁷⁷ Lu、 ⁹⁰ Y is Y ²²⁵ At least one of Ac.

The nuclide is bound to a nuclide-labelling group R in a compound of formula (I) ₁ Chelation is performed.

The nuclide labelGroup R ₁ Introduction of nuclides by the following band-nuclide structure ^{123/124/125/131} I or ¹⁸ F，

The nuclide labeling group R ₁ Selected from any one of the following structures:

in the present invention, when R in formula (I) ₁ The following structures are adopted respectively:

the VISTA compounds were DNCA, NNCA, DCA, NCA, HCA, respectively, and the structures thereof were as follows:

the preparation method of the VISTA targeted probe adopts a wet method or a freeze-drying method for preparation.

The wet method comprises the following steps: dissolving a compound shown in a formula (1) in buffer solution or deionized water, adding a solution containing the radionuclide, reacting for 10-30 min at the temperature of 100 ℃ at room temperature, diluting with normal saline or water for injection, and filtering with a sterile filter membrane to obtain the radionuclide-labeled complex injection.

The freeze-drying method comprises the following steps: dissolving a compound of the formula (1) in buffer solution or deionized water, subpackaging in a freeze-drying container, freeze-drying, sealing to obtain a freeze-drying medicine box, and adding related excipients, antioxidants or acid-base regulators into the freeze-drying medicine box according to the requirement; adding deionized water or buffer solution into the freeze-dried medicine box for dissolution, adding the solution containing the radionuclide, reacting for 10-30 min at the room temperature to 100 ℃, diluting with normal saline or water for injection, and filtering with a sterile filter membrane to obtain the radionuclide-labeled complex injection.

Application of the VISTA targeted probe in preparation of products for detecting or treating diseases or symptoms related to the VISTA; the VISTA targeted probe is prepared into injection and is administrated by intravenous injection; such conditions include tumors or autoimmune diseases; the tumor comprises breast cancer, ovarian cancer, lung cancer, colorectal cancer, prostate cancer, lung cancer, fibrosarcoma, bone and connective tissue sarcoma, renal cell carcinoma, gastric cancer, pancreatic cancer or skin melanoma, etc.; the autoimmune diseases include lupus erythematosus, rheumatoid arthritis, hepatitis, autoimmune encephalomyelitis, scleroderma, psoriasis, sjogren's syndrome, polyarteritis nodosa, asthma, etc.; imaging diagnostic modalities include Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET). The treatment may include nuclide targeted therapy, immunotherapy, or combination therapy.

Compared with the prior art, the technical scheme of the invention has the beneficial effects that:

the probe provided by the invention is an important supplement of accurate immunology, fills the blank in the field of VISTA nuclide targeting probes, is suitable for in-vivo screening of immune microbial environment biomarkers for diseases such as tumors, avoids molecular pathological detection of in-vitro specimens, and provides an effective new way for visual diagnosis, identification, monitoring, treatment and prognosis evaluation of serious diseases such as tumors. The method is more beneficial to constructing an 'inflammatory-cancer transformation system and an immune relation network' around a VISTA target spot, and explores a treatment mechanism of tumors and related diseases based on the 'inflammatory-cancer transformation system and the immune relation network'. Biological data show that the probe has the advantages of strong targeting property, high target/non-target ratio and the like, and is favorable for the commercialized application and clinical popularization of the probe.

Drawings

FIG. 1 is a mass spectrum of Compound NNCA.

Fig. 2 is a mass spectrum of compound DNCA.

FIG. 3 is a mass spectrum of compound HCA.

FIG. 4 is an HPLC chromatogram of Compound NNCA.

Fig. 5 is an HPLC profile of compound DNCA.

FIG. 6 is an HPLC chromatogram of Compound HCA.

FIG. 7 is a complex ⁶⁸ HPLC profile of Ga-DNCA and stable results in physiological saline.

FIG. 8 is a complex ^99m HPLC profile of Tc-HCA and stability in physiological saline results.

FIG. 9 is a diagram of ⁶⁸ MicroPET imaging of Ga-DNCA in B16 melanoma-bearing, CT26 colon cancer, 4T1 breast cancer, and PANC02 pancreatic cancer mice for 0.5 h.

Detailed Description

In order to make the technical problems, technical schemes and beneficial effects to be solved more clear and obvious, the invention is further described in detail below with reference to the accompanying drawings and embodiments. Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.

The structural formula of the FAPI compound is shown as the following (I):

wherein: r is R ₁ Is a nuclide labeling group; nuclides include ¹⁷⁷ Lu、 ⁹⁰ Y、 ¹⁸ F、 ^99m Tc、 ⁶⁴ Cu、 ⁶⁸ Ga、 ²²⁵ Ac、 ⁸⁹ Zr、 ⁶² Cu、 ⁶⁷ Cu、 ⁶⁴ Gd、 ⁸⁶ Y、 ⁸⁹ Sr， ¹⁵³ Sm、 ¹⁴⁹ Tb、 ¹⁶¹ Tb、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ²¹² Pb、 ²¹³ Bi、 ²²³ Ra、 ²²⁶ Th、 ²²⁷ Th、 ^123/124/125/ ¹³¹ I、 ²¹¹ At or ¹¹¹ At least one of In, preferably ⁶⁸ Ga、 ^99m Tc、 ¹⁸ F、 ¹⁷⁷ Lu、 ⁹⁰ Y is Y ²²⁵ At least one of Ac.

The nuclide targets the nuclide labeling group R in the compound structure through VISTA ₁ Chelation is performed.

For metal ions, the radiolabeled probe may be prepared by wet or freeze drying a radionuclide-containing compound with a compound of formula (I).

The wet method comprises the following steps: and dissolving the VISTA target compound in buffer solution or deionized water, adding a solution containing the radionuclide, reacting for 10-30 min at the temperature of 100 ℃, diluting with normal saline or water for injection, and filtering with a sterile filter membrane to obtain the radionuclide-labeled complex injection.

The freeze-drying method comprises the following steps: dissolving the VISTA target compound in buffer solution or deionized water, subpackaging in a freeze-drying container, freeze-drying, sealing to obtain a freeze-drying medicine box, and adding related excipients, antioxidants or acid-base regulators into the freeze-drying medicine box according to the requirement; adding deionized water or buffer solution into the freeze-dried medicine box for dissolution, adding the solution containing the radionuclide, reacting for 10-30 min at the room temperature to 100 ℃, diluting with normal saline or water for injection, and filtering with a sterile filter membrane to obtain the radionuclide-labeled complex injection.

Specifically, the container for sub-packaging in the lyophilization is preferably a cryopreservation tube or a tube antibiotic bottle, and an excipient or an antioxidant such as mannitol, ascorbic acid and the like can be added into the kit according to the molding condition of the lyophilized powder of the kit, and the molding of the kit can be optimized by adjusting the dosage of the VISTA targeting compound and the excipient. The buffer solution is used for stabilizing the pH value of the reaction solution, and can be acetate, lactate, tartrate, malate, maleate, succinate, ascorbate, carbonate, phosphate, a mixture thereof and the like.

If the labeling rate and radiochemical purity are low, the invention provides a preferred purification mode comprising: a Sep-Pak C18 separation column was taken and activated by 10mL absolute ethanol followed by 10mL water. Diluting the reaction solution with water, separating and purifying by Sep-Pak C18 chromatographic column, washing the chromatographic column with buffer solution or water to remove unreacted radioactive ions, eluting with ethanol solution to obtain radionuclide-labeled complex, removing organic solvent by nitrogen blowing, diluting with normal saline or water for injection, and aseptically filtering to obtain injection of radionuclide-labeled complex with high radiochemical purity.

Example 1:

the synthesis of precursor compounds DNCA or HCA or DCA or NCA based on the structure of formula (I) comprises the steps of:

dissolving p-SCN-Bn-NOTA or p-NCS-Bz-DOTA-GA or HYNIC-NHS or DOTA-NHS or NOTA-NHS and the compound 1 in DMSO or DMF solution, adding a proper amount of DIPEA, and reacting for 4-12 hours at room temperature. The product was purified by HPLC, the target fraction was collected and lyophilized to give the corresponding compound NNCA, DNCA, HCA, DCA or NCA. The target product was confirmed by mass spectrometry. Mass spectrometry spectra and HPLC spectra of the compounds NNCA, DNCA or HCA are shown in fig. 1 to 6.

The other preparation methods of VISTA target compounds based on the structure (I) can refer to the synthesis process, and the difference is that the corresponding nuclide labeling groups are replaced, so that the corresponding compound structure can be obtained.

The synthetic routes for precursor compounds DNCA or HCA or DCA or NCA based on the structure of formula (I) in this example are shown below:

example 2:

⁶⁸ the Ga nuclide labeling process is as follows:

wet process: will be about 37 to 3700MBq ⁶⁸ GaCl ₃ Hydrochloric acid solution (eluted from germanium gallium generator) is added into a centrifuge tube containing 0.5-2 mL of acetic acid-acetate solution of DNCA (20-200 mug) prepared in example 2, and the mixture is placed at room temperature to 100 ℃ for reaction for 20 minutes, cooled to room temperature and diluted by normal saline or water for injectionAnd sterile filtering to obtain the marked compound injection.

And (3) lyophilization: an amount of buffer and about 37 to 3700MBq ⁶⁸ GaCl ₃ Adding hydrochloric acid solution (leached from a germanium gallium generator) into a freeze-dried medicine box containing DNCA (20-200 mu g), uniformly mixing and dissolving, placing the mixture at room temperature to 100 ℃ for reaction for 20min, cooling to room temperature, diluting with normal saline or water for injection, and carrying out sterile filtration to obtain the marked compound injection.

If the radiochemical purity is lower than 95%, purification is needed, and the purification steps are as follows: a Sep-Pak C18 separation column was taken and activated by 10mL absolute ethanol followed by 10mL water. After diluting the labeling solution with 10mL of water, the mixture was applied to a separation column. Washing the separation column with water to remove unreacted ⁶⁸ Ga ion is leached by ethanol solution to obtain ⁶⁸ Ga-labeled complexes. Removing the organic solvent by nitrogen blowing, diluting with normal saline, and aseptically filtering to obtain the labeling compound injection.

As shown in FIG. 7, for the labeled compounds ⁶⁸ The Ga-DNCA sample is analyzed and identified by HPLC, and the radiochemical purity is more than 95%.

Example 3:

^99m tc nuclide labeling: by SnCl ₂ N-tris (hydroxymethyl) methylglycine (Tricine) and triphenylphosphine-sodium trimetaphosphate (TPPTS) as co-ligands ^99m Labeling of Tc.

Wet process: freshly prepared SnCl ₂ Solution (SnCl) ₂ 20 mu L of the solution containing 20 to 200 mu g of HCA compound, 1 to 50mg of Tricine and 1 to 10mg of TPPTS, and then immediately adding 37 to 7400MBq of freshly leached Na ^99m TcO ₄ Eluting the eluent (eluting from a molybdenum technetium generator), uniformly mixing, sealing by a gland, reacting at room temperature to 100 ℃ for 30 minutes, cooling to room temperature, diluting with normal saline or water for injection, and carrying out sterile filtration to obtain the marked compound injection.

And (3) lyophilization: fresh Na of about 37 to 3700MBq ^99m TcO ₄ The eluate (eluted from the molybdenum technetium generator) was added to a solution containing 20-200. Mu.g of HCA compound, 1. Mu.g50mg Tricine and 1-10 mg TPPTS (containing mannitol and ascorbic acid), mixing, sealing with a gland, reacting at room temperature to 100deg.C for 30min, cooling to room temperature, diluting with physiological saline or water for injection, and sterile filtering to obtain the marked compound injection.

If the radiochemical purity is lower than 95%, purification is needed, and the purification steps are as follows: a Sep-Pak C18 separation column was taken and activated by 10mL absolute ethanol followed by 10mL water. After diluting the labeling solution with 10mL of water, the mixture was applied to a separation column. Washing the separation column with water to remove unreacted ^99m TcO ₄ ^- Eluting with ethanol solution to obtain ^99m Tc-labeled complexes. Removing organic solvent by nitrogen blowing instrument, diluting with physiological saline, and aseptic filtering to obtain the labeled compound ^99m Tc-HCA injection.

As shown in FIG. 8, for the labeled compounds ^99m Tc-HCA samples were analyzed by HPLC to identify radiochemical purity greater than 95%.

Example 4:

1. lipid water partition coefficient (log P) determination

100 mu L of diluted solution ^99m Tc-HCA or ⁶⁸ Ga-DACA was added to a centrifuge tube containing a mixture of 2.9mL PBS and 3mL n-octanol, centrifuged at 6000rpm for 5min after vortexing for 3min, 100. Mu.L of liquid was taken from each of the aqueous and n-octanol phases and counted by gamma-counter radioactivity. The experiment was repeated three times to average. The calculation formula of log P is:

P＝I _{organic phase} /I _{Aqueous phase}

Wherein I is _{Organic phase} Representing the measured radioactivity count, I, in the organic phase _{Aqueous phase} Representing the radioactivity count measured in the aqueous phase. The lipid-water distribution coefficient of each radiolabeled targeting probe was finally determined by calculation. Measured labeling compounds ^99m Tc-HCA ⁶⁸ The Ga-DACA has log P values of-2.76 and-2.63, respectively, and is water-soluble.

2. In vitro stability test

Labeling compound to be dissolved in physiological saline ⁶⁸ Ga-DNCA or ^99m Tc-HCA at room temperatureThe samples were placed under them for various times and analyzed by HPLC. At the time point tested, the probe remained radiochemically pure>95% shows that the product has stable property in the appointed solution and is not easy to decompose. ⁶⁸ Ga-DNCA ^99m The results of HPLC identification of the stability of Tc-HCA physiological saline are shown in figures 7 and 8, which show that the Tc-HCA physiological saline maintains higher stability in the system>95％)。

Example 5:

tumor model mouse MicroPET imaging

A radiochemical purity of greater than 95% was prepared as in example 2 ⁶⁸ Ga-DNCA labeled complex solution, take 0.2mL (about 11 MBq) through loading B16 melanoma, CT26 colon cancer, 4T1 breast cancer and PANC02 pancreatic cancer mouse tail vein injection, at 30 minutes after injection time point of MicroPET imaging. As shown in FIG. 9, the area indicated by the arrow is the tumor position, the present invention ⁶⁸ Ga-labeled probe ⁶⁸ Ga-DNCA has obvious enrichment and retention in mouse tumors, has fast removal in normal tissues and organs such as muscles, livers, blood pools and the like, and has high target-to-non-target ratio. The bladder location has a higher radioactivity signal, which means that the metabolism of the probe in vivo is excreted outside the body via urine.

Claims

1. A VISTA targeted probe, characterized by: the probe is based on a VISTA compound, and the structure of the VISTA compound is shown as the following formula (1):

wherein R is ₁ Is a nuclide labeling group selected from any one of the following structures:

the nuclide is selected from ¹⁷⁷ Lu、 ⁹⁰ Y、 ⁶⁴ Cu、 ⁶⁸ Ga、 ⁸⁹ Zr、 ^99m Tc、 ¹⁸⁸ Re、 ²¹³ Bi、 ²²³ Ra、 ²²⁵ Ac、 ²¹¹ At or ¹¹¹ At least one of In.

2. The VISTA targeted probe of claim 1, wherein: the nuclide is selected from ⁶⁸ Ga、 ^99m Tc、 ¹⁷⁷ Lu、 ⁹⁰ Y is Y ²²⁵ At least one of Ac.

3. The VISTA targeted probe of claim 1, wherein: the nuclide is bound to a nuclide-labelling group R in a compound of formula (I) ₁ Chelation is performed.

4. A method for preparing a VISTA targeted probe according to any one of claims 1 to 3, characterized in that: the preparation is carried out by adopting a wet method or a freeze-drying method.

5. The method of claim 4, wherein the wet process comprises the steps of: dissolving a compound shown in a formula (1) in buffer solution or deionized water, adding a solution containing the radionuclide, reacting for 10-30 min at the temperature of 100 ℃ at room temperature, diluting with normal saline or water for injection, and filtering with a sterile filter membrane to obtain the radionuclide-labeled complex injection.

6. The method of claim 4, wherein the lyophilization process comprises the steps of: dissolving a compound of the formula (1) in buffer solution or deionized water, subpackaging in a freeze-drying container, freeze-drying, sealing to obtain a freeze-drying medicine box, adding related excipients, antioxidants or acid-base modifier into the freeze-drying medicine box according to the requirement, wherein the related excipients adopt mannitol, the antioxidants adopt ascorbic acid, and the acid-base modifier adopts at least one of acetate, lactate, tartrate, malate, maleate, succinate, ascorbate, carbonate and phosphate; adding deionized water or buffer solution into the freeze-dried medicine box for dissolution, adding the solution containing the radionuclide, reacting for 10-30 min at the room temperature to 100 ℃, diluting with normal saline or water for injection, and filtering with a sterile filter membrane to obtain the radionuclide-labeled complex injection.

7. Use of the VISTA targeted probe of any one of claims 1 to 3 or the VISTA targeted probe prepared by the preparation method of any one of claims 4 to 6, characterized in that: the application in preparing products for detecting diseases or symptoms related to VISTA; the VISTA targeted probe is prepared into injection and is administrated by intravenous injection; such conditions include tumors or autoimmune diseases; the tumor comprises breast cancer, ovarian cancer, lung cancer, colorectal cancer, prostate cancer, fibrosarcoma, bone and connective tissue sarcoma, renal cell carcinoma, gastric cancer, pancreatic cancer or skin melanoma; the autoimmune diseases include lupus erythematosus, rheumatoid arthritis, hepatitis, autoimmune encephalomyelitis, scleroderma, psoriasis, sjogren's syndrome, polyarteritis nodosa, asthma.