Liquid touching neuron specific tracing imaging agent, preparation method and application

Abstract

The invention relates to a touch fluid neuron specific tracing imaging agent, a preparation method and application, and belongs to the technical field of nuclear medicine imaging, the tracing imaging agent is composed of a nerve tracer agent horseradish peroxidase (HRP), zirconium-89 isotope chelated by deferoxamine and a pharmaceutically acceptable carrier, the effective tracking window of the tracer agent is prolonged to 120 hours, and the effective tracking window of the tracer agent is prolonged to 120 hours. The multi-stage synaptic transfer period of the fluid touching neuron can be completely covered; the spatial resolution is improved by 5-8 times compared with that of traditional FDG-PET, and a fluid touching neuron cluster can be accurately presented; the in-vitro serum stability is excellent, the radiochemical purity is still larger than or equal to 80.5% within 120 hours, PET / MRI bimodal imaging can be adapted, drug administration is conducted through lateral ventricle minimally invasive injection, the probe is suitable for various animal models such as mice, rats and non-human primates, in-vivo dynamic tracing of sympathetic fluid neuron trans-synapses can be achieved on the premise that no tissue damage exists, and the probe has a good application prospect. And a key technical tool is provided for neural circuit analysis, neuroscience basic research and preclinical research of neurodegenerative diseases.

Description

Technical Field

[0001] This invention relates to the field of nuclear medicine imaging technology, specifically to a fluid-sensitive neuron-specific tracer imaging agent, its preparation method, and its application. Background Technology

[0002] In neuroscience research, neuronal tracing techniques are crucial for elucidating the structure and function of neural networks. Currently widely used fluorescent marker tracking techniques have several limitations: First, limited resolution makes it difficult to clearly observe the fine structures of individual neurons (such as synapses and neurites); second, insufficient fluorescence penetration depth prevents effective tracking of deep neurons; third, poor stability of fluorescent markers, which degrade easily during long-term experiments, leading to signal attenuation; fourth, the need for physical or chemical damage to neurons to introduce the markers, potentially altering normal neuronal function; fifth, the ability to track only single or grouped neurons, making it impossible to observe the activity of the entire neural network; and sixth, fluorescence visualization is primarily observed on tissue sections from euthanized animals, not in live animals.

[0003] Traditional radiolabeling methods also have drawbacks: the use of short-half-life nuclides results in short tracking times, requiring increased nuclide doses to extend tracking time, thus increasing radiation doses; the labeling chemical reaction conditions are harsh (e.g., high temperatures), the methods are complex, technically challenging, and the imaging results are poor. Taking the commonly used chelating agent DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) as an example, its binding to 89Zr requires a high temperature of 40-50℃ and a long reaction time of 30-60 minutes, which may affect protein activity. Furthermore, DOTA's affinity for 89Zr is weaker than that of DFO, requiring optimization of pH, buffer systems, and other conditions to improve the labeling rate. Additionally, DOTA's macrocyclic structure may limit coupling sites with some proteins, affecting targeting. Moreover, traditional autoradiographic substances have relatively small molecular weights, and when administered via the ventricular system, they often cross the brain barrier and diffuse freely, making it difficult to precisely target specific functional nuclei within the brain.

[0004] HRP (horseradish peroxidase, molecular weight approximately 40 kDa) possesses transsynaptic properties, but its application in tracking presents significant bottlenecks: its diffusion rate in living tissue is slow, and transsynaptic transmission requires penetrating the synaptic cleft and entering the next level of neurons, a process that is extremely inefficient in living environments, making it difficult to achieve real-time tracking of long-distance, multi-level circuits; HRP detection relies on substrate (such as DAB) catalytic colorimetric reactions, which must be carried out in fixed, permeabilized ex vivo tissues, making real-time imaging impossible in living conditions; the HRP signal intensity decreases significantly after each level of synaptic transmission, and the signal may fall below the detection threshold after crossing multiple synapses, making it difficult to fully depict long-range pathways. Its molecular characteristics fundamentally conflict with the requirements of real-time in vivo imaging. Summary of the Invention

[0005] The present invention aims to solve the above-mentioned technical problems by providing a specific tracer imaging agent for tactile neurons, its preparation method, and its application.

[0006] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0007] A tactile neuron-specific tracer imaging agent comprising horseradish peroxidase, a zirconium-89 isotope chelated by deferoxamine, and a pharmaceutically acceptable carrier.

[0008] Preferably, the deferoxamine and horseradish peroxidase achieve specific binding through C-terminal modification.

[0009] Preferably, the specific activity of the tracer imaging agent is in the range of 18.5-74 MBq / mg.

[0010] Preferably, the pharmaceutically acceptable carrier is selected from at least one of HEPES buffer, Na2CO3 / NaHCO3 buffer, and acetate / sodium acetate buffer.

[0011] Preferably, the radiochemical purity of the tracer in serum is as follows, as determined by radiometric thin-layer chromatography: 91.5±0.56% at 24 hours, 89.5±0.63% at 48 hours, and 80.5±1.24% at 120 hours.

[0012] The preferred horseradish peroxidase molecules are large and cannot freely cross the cerebrospinal fluid-brain barrier to enter brain tissue. They do not mark non-fluid-contact neurons in the brain, but can only specifically mark neurons whose terminals extend into the cerebrospinal fluid (i.e., fluid-contact neurons).

[0013] This invention also provides a method for preparing a hygroscopic neuron-specific tracer imaging agent, comprising the following steps:

[0014] S1: Preparation of DFO-HRP solution:

[0015] (1) Dissolve ferricamine in DMSO to prepare a DFO solution with a concentration of 20 μg / μl; dissolve horseradish peroxidase in ultrapure water to prepare an HRP solution with a concentration of 10 μg / μl;

[0016] (2) Take HRP solution and add 0.1 mol / L Na2CO3 / NaHCO3 buffer solution, pH=9.0, and add 0.1 mol / L Na2CO3 solution dropwise to adjust the pH of the system to 9.0;

[0017] (3) According to the molar ratio of DFO to HRP of 9:1, DFO solution was added to the above HRP solution in 3 portions, with an interval of 10 min between each portion, and the reaction was carried out in a nitrogen blower at 37℃ for 120 min.

[0018] (4) The reaction products were sequentially purified by PD-10 desalting column separation and 100×10 3 The ultrafiltration tube was concentrated and washed twice to collect the DFO-HRP solution.

[0019] S2: 89 Preparation of Zr-DFO-HRP:

[0020] (1) Take 89 Zr was added to 9 volumes of 0.1 mol / L HEPES buffer, and the pH of the system was adjusted to 7 by adding 1 mol / L Na₂CO₃ solution. 89 Zr solution;

[0021] (2) 89 Zr solution was added to DFO-HRP solution and reacted at 37°C for 15 min to obtain a tracer imaging agent.

[0022] The present invention also provides the application of a liquid-contact neuron-specific tracer imaging agent in in vivo transsynaptic imaging of neurons for non-therapeutic purposes.

[0023] Preferably, the in vivo imaging is PET / MRI dual-modal imaging.

[0024] Preferably, the tracer imaging agent in the application is administered via lateral ventricle injection to perform long-range dynamic tracing of multi-level neuronal circuits, with an effective tracking window of 6-120 hours.

[0025] By using the above methods and reagents, the present invention has the following advantages:

[0026] 1. Design of a bifunctional chelating agent: DFO (deferroamine) is selected as a bifunctional chelating agent. Its amino group at one end can specifically couple with the carboxyl group (C-terminus) of HRP via an amide bond, while its hydroxamic acid group at the other end can... 89 Zr forms a stable six-membered ring chelate, solving the problems of low labeling efficiency, poor stability, and damage to protein activity caused by traditional chelating agents (such as DOTA); and DFO has a small molecular weight (about 560 Da) and low steric hindrance, so it does not affect the transsynaptic activity of HRP and the uptake capacity of lyophilized neurons.

[0027] 2. pH-sensitive labeling system: Establish a staged pH precision control strategy—DFO and HRP coupling stage (pH=9.0), maintain an alkaline environment through Na2CO3 / NaHCO3 buffer to promote amide bond formation; 89 During the Zr labeling stage (pH = 7.0), the pH was adjusted to neutral using HEPES buffer to avoid the degradation of HRP activity by extreme pH, while ensuring... 89 The chelation reaction between Zr and DFO proceeds efficiently (reaction efficiency ≥ 95%).

[0028] 3. Neuronal targeting optimization: By controlling the molar ratio of DFO to HRP (9:1), reaction temperature (37℃), and time (120min), the activity retention rate of HRP is maximized (≥90%). Combined with intraventricular injection, the tracer imaging agent is directly delivered to the central nervous system, reducing non-specific uptake by peripheral tissues (such as liver and kidneys) and improving the specific uptake efficiency of hygroscopic neurons (10-15 times higher than intravenous injection).

[0029] 4. HRP and 89 Zr chelated with DFO to obtain a specific tracer and contrast agent for touch neurons combines horseradish peroxidase-specific labeling of touch neurons and 89 Zr offers the dual advantages of non-specific, high-quality imaging: specificity of targeted labeling, timeliness of the observation window, clarity of tracer imaging, safety for biological metabolism, and simplicity of preparation methods.

[0030] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the invention will become readily apparent from the full text and the following detailed description. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 This is a schematic diagram of the molecular formula reaction of the present invention.

[0033] Figure 2 This is a triaxial PET-CT image of the experimental mice of this invention after 1 hour of injection of a tracer imaging agent.

[0034] Figure 3 This is a triaxial PET-CT image of the experimental mice of this invention 12 hours after injection of the tracer imaging agent.

[0035] Figure 4 This is a PET-CT image of the experimental mice of this invention taken 24 hours after injection of a tracer imaging agent.

[0036] Figure 5 This is a triaxial PET-CT image of the experimental mice of this invention 48 hours after injection of the tracer imaging agent.

[0037] Figure 6This is a triaxial PET-CT image of the experimental mice of this invention 72 hours after injection of the tracer imaging agent.

[0038] Figure 7 This is a triaxial PET-CT image of the experimental mice of this invention 96 hours after injection of the tracer imaging agent.

[0039] Figure 8 This is a triaxial PET-CT image of the experimental mice of this invention 120 hours after injection of the tracer imaging agent. Detailed Implementation

[0040] Specific embodiments of the invention will now be described in detail. Although the invention is described in conjunction with these specific embodiments, it should be understood that the invention is not intended to be limited to these specific embodiments. Rather, these embodiments are intended to cover alternative, modified, or equivalent embodiments that may be included within the spirit and scope of the invention as defined by the claims. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the invention. The invention may be practiced without some or all of these specific details. In other instances, well-known processes have not been described in detail so as not to unnecessarily obscure the invention.

[0041] When used in conjunction with the terms "comprising," "method comprising," or similar language in this specification and appended claims, the singular forms "a," "some," and "the" include plural references unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0042] The present invention will now be described in further detail with reference to the full text.

[0043] In conjunction with the full text and the appendix Figure 1 As shown, the tactile neuron-specific tracer imaging agent of the present invention mainly consists of three parts:

[0044] Functional protein component: Horseradish peroxidase (HRP), which utilizes its inherent non-synaptic properties to provide a targeting basis for transsynaptic tracking of fluid-contact neurons.

[0045] Radiolabeled component: Zirconium-89 chelated via deferoxamine (DFO) 89 Zr isotopes, 89 Zr has a suitable half-life (approximately 78.4 hours) to meet the requirements of long-term tracking, and its decay characteristics are suitable for PET imaging, providing high-resolution imaging signals.

[0046] Pharmaceutical carrier components: Pharmaceutically acceptable carriers, including HEPES buffer (0.1 mol / L), Na2CO3 / NaHCO3 buffer (0.1 mol / L), etc., are used to maintain the stability of the tracer and imaging agent, adjust the pH of the reaction system, and ensure the stability of the physicochemical properties of the tracer and imaging agent during preparation and in vivo application.

[0047] Maintain the pH stability of the tracer imaging agent (pH=9.0 during coupling phase and pH=7.0 during labeling phase); provide an isotonic environment to prevent aggregation of the tracer imaging agent during preparation and storage; promote the biocompatibility of the tracer imaging agent in vivo and reduce immune responses.

[0048] The preparation method of the tactile neuron-specific tracer imaging agent of the present invention specifically includes the following steps:

[0049] S1: Preparation of DFO-HRP solution:

[0050] (1) Weigh 3 mg of DFO (deferroamine), dissolve it in 150 μL of dimethyl sulfoxide (DMSO), mix thoroughly to obtain a DFO solution with a concentration of 20 μg / μl;

[0051] Weigh 20 mg of HRP (horseradish peroxidase), dissolve it in 2000 μL of ultrapure water, and obtain an HRP solution with a concentration of 10 μg / μl.

[0052] (2) pH adjustment: Take 10 1.5ml test tubes, add 200μL (containing 200μgHRP) of the above HRP solution to each test tube, then add 1ml of 0.1mol / L Na2CO3 / NaHCO3 buffer solution, and then add 0.1mol / L Na2CO3 solution dropwise. Use a pH meter to monitor in real time and adjust the pH value of the solution to about 9.0 for later use.

[0053] (3) Coupling reaction: Calculate the required volume of DFO solution according to the molar ratio of DFO to HRP of 9:1 (11.2 μL DFO solution is required for each test tube). Add the DFO solution slowly to the above HRP solution in 3 portions, with an interval of 10 min between each addition, to ensure that DFO and HRP are fully in contact and react. Place the test tube in a nitrogen blower at 37℃ and react for 120 min to complete the coupling of DFO and HRP.

[0054] (4) Purification process:

[0055] After the reaction is complete, the reaction solution is transferred to a PD-10 desalting column and ultrapure water is used as the eluent for separation and purification to remove unreacted free DFO and other impurities.

[0056] Collect the desalted DFO-HRP solution and use 100×103 The ultrafiltration tube is concentrated and washed twice to further remove small molecule impurities and obtain a purified DFO-HRP solution. If the volume obtained after desalting is sufficient for subsequent injection, the ultrafiltration concentration step can be omitted.

[0057] S2: 89 Preparation and quality control of Zr-DFO-HRP (buffer system, reaction time, purification method)

[0058] Pretreatment of 89Zr solution: Take 37 MBq of zirconium oxalate 89Zr solution (approximately 100 μL) and place it in a reaction flask. Add 9 times the volume (900 μL) of 0.1 mol / L HEPES buffer and mix thoroughly. Then, slowly add 1 mol / L Na₂CO₃ solution (pH = 10) dropwise, monitoring the pH with a pH meter as you add. Adjust the pH of the reaction system to 7.0 to obtain a solution with suitable pH. 89 Zr mixed solution.

[0059] Labeling reaction: The pH adjusted above 89 The entire Zr mixed solution was added to the DFO-HRP solution obtained in step 1, and the mixture was placed in a 37°C constant temperature water bath for 15 minutes, with gentle shaking every 5 minutes to ensure... 89 Zr and DFO are fully chelated, and the reaction yields... 89 Zr-DFO-HRP solution (i.e., a specific tracer and imaging agent for target hygroscopic neurons).

[0060] Horseradish peroxidase (HRP) has a molecular weight of 40 kDa and can diffuse freely in cerebrospinal fluid, but it cannot cross the cerebrospinal fluid-brain barrier to enter brain tissue. Therefore, when administered via the lateral ventricle, it can only be pinocytotically labeled by cerebrospinal fluid-contacting neurons, retrogradely marking the entirety of these neurons. Because of its natural non-synaptic transmission property, it will not cross-synapse to label other non-cerebrospinal neurons, thus forming the biological basis and reliable localization principle for targeted tracing of cerebrospinal neurons.

[0061] Because HRP can be retrogradely entered into cells via pinocytosis at the terminal synovial neurons in the cerebrospinal fluid, and then penetrate the synaptic cleft to enter the next level of neurons, it provides a targeting basis for tracking. 89 Zr is a positron-emitting nuclide (positron energy 0.903 MeV) with a half-life of 78.4 hours, which can meet the requirements for long-term imaging up to 120 hours; DFO and 89 Zr chelation is stable, with a radiochemical purity of ≥89.5% in serum after 48 hours in vitro, avoiding free Zr. 89 Zr interference with imaging results.

[0062] Sample preparation: Take 1.85-3.70 MBq (approximately 200 μL). 89Place the Zr-DFO-HRP solution in a centrifuge tube, add 500 μL of human serum or a suitable solvent (such as HEPES buffer), mix thoroughly, and then store in a lead container at room temperature for subsequent labeling rate determination and stability testing.

[0063] Labeling rate determination: Take a small amount using a capillary tube 89 The Zr-DFO-HRP solution was spotted 1 cm below the bottom of the chromatographic paper, placed in 0.5 mol / L citric acid solution, and then removed and placed on the chromatograph to determine the product labeling rate.

[0064] In vivo metabolic regulation technology of tracer imaging agents (blood-brain barrier penetration enhancement method):

[0065] By optimizing the injection method of the tracer (such as intraventricular injection), the tracer can be directed to label only sympathetic neurons, reducing the uptake of the tracer by peripheral tissues and other brain tissues, and increasing the concentration of the tracer in sympathetic neurons; simultaneously, by utilizing the non-transsynaptic properties of HRP and 89 Zr's long half-life promotes the distribution of tracers, ensuring the achievement of specific, long-term, high-resolution, safe and harmless in vivo imaging.

[0066] Quality control methods:

[0067] Labeling rate determination: The labeling rate of 89Zr-DFO-HRP was determined using radioactive thin-layer chromatography (R-TLC). The specific procedure is as follows: A small amount of 89Zr-DFO-HRP solution was drawn into a capillary tube and spotted 1 cm below the bottom of the chromatographic paper (avoiding excessively large or concentrated spots). After the sample was allowed to air dry, the chromatographic paper was placed in 0.5 mol / L citric acid solution for development at a distance of 10-15 cm. After development, the chromatographic paper was removed and dried, then placed on the chromatograph for radioactivity detection. The product labeling rate was calculated based on the position and intensity of the radioactive peaks. If the labeling rate is lower than expected, further purification using a PD-10 desalting column should be considered to remove free radicals. 89 Zr.

[0068] Stability determination: The R-TLC method was also used for detection. 89 The labeling rate of Zr-DFO-HRP at different time points (6h, 24h, 48h, 72h, 96h, 120h, 144h)

[0069] To assess the stability of the tracer imaging agent. It will be placed in serum or a suitable solvent. 89 Zr-DFO-HRP samples were stored at room temperature, and samples were taken at the above-mentioned time points. The labeling rate was determined according to the above-mentioned method, and the radiochemical purity at each time point was recorded. The experimental results showed... 89Zr-DFO-HRP exhibits good stability in in vitro serum, with a radiochemical purity of 91.5±0.56% at 24 hours, 89.5±0.63% at 48 hours, and 80.5±1.24% at 120 hours, which meets the requirements for in vivo imaging within 120 hours.

[0070] Example:

[0071] To verify the performance of the tracer imaging agent of the present invention, the following embodiments were designed, including animal model comparison experiments, dynamic imaging time window verification, synaptic cascade tracer ability test and label stability test.

[0072] I. Experimental Materials:

[0073] Experimental animals: 4 SPF-grade mice (weighing 20-25g, half male and half female), and several non-human primates (such as rhesus monkeys, weighing 3-5kg) (for further verification).

[0074] Experimental reagents: those prepared in this invention 89 Zr-DFO-HRP tracer imaging agent (specific activity 18.5-74 MBq / mg), 89 Zr solution (zirconium oxalate) 89 Zr, 1mCi), mouse anesthetic (isoflurane).

[0075] Experimental equipment: PET / CT scanner, pH meter, nitrogen evaporator, PD-10 desalination column, 100×10 3 Ultrafiltration tubes, radioactive thin-layer chromatograph, lead container.

[0076] II. Animal Model Comparative Experiments

[0077] Grouping: Four mice were divided into a control group (1 mouse) and an experimental group (3 mice).

[0078] Injection procedure:

[0079] Control group: Mice were anesthetized with isoflurane and injected via lateral ventricle at a dose of 10 μCi / 10 μL. 89 Zr solution.

[0080] Experimental group: Three mice were anesthetized with isoflurane and injected with 10 μCi / 10 μL via lateral ventricle injection. 89 Zr-DFO-HRP tracer imaging agent.

[0081] Imaging detection: The injection time and syringe activity of each mouse were recorded. PET / CT imaging was performed immediately after injection, and at 1h, 4h, 12h, 1d, 2d, 3d, 4d, 5d, 6d, and 7d. The imaging of the mouse brain and various tissues throughout the body was observed and recorded.

[0082] Results Analysis: In the control group, weak imaging of the brain was observed only in the initial stage after injection, and the signal rapidly spread throughout the body, especially to metabolic organs such as the liver. After 12 hours, there was virtually no obvious signal in the brain. In the experimental group, clear lateral ventricle imaging was observed in the brain of mice 1 hour after injection, and the signal persisted in the brain for a longer period. Even after 120 hours (5 days), there was still a significant drug signal in the lateral ventricle. The uptake in the liver and surrounding soft tissues peaked at 2 days and then gradually weakened, indicating that the present invention... 89 Zr-DFO-HRP tracer imaging agent exhibits good retention and targeting in the brain, superior to simple [methods]. 89 Zr solution.

[0083] III. Dynamic Imaging Time Window Verification:

[0084] Experimental procedure: Select the mouse with the best imaging results from the experimental group and monitor its performance closely after injection of 370 kBq. 89 The changes in radioactivity distribution in key areas such as the lateral ventricle and liver after Zr-DFO-HRP tracer imaging agent were investigated. The radioactivity (unit: kBq / ml) of the region of interest (lateral ventricle and liver) was measured at 1h, 24h, 48h, 72h, 96h and 120h after injection.

[0085] Results record (e.g.) Figures 2-8 As shown):

[0086] 1 hour: The radioactivity in the lateral ventricle was 357.95 kBq / ml, and no obvious radioactive signal was detected in the liver.

[0087] 24h: The radioactivity of the lateral ventricles was 96.81 kBq / ml, and the radioactivity of the liver was 2.23 kBq / ml.

[0088] 48h: The radioactivity in the lateral ventricles was 68.27 kBq / ml, and the radioactivity in the liver was 2.63 kBq / ml (reaching its peak).

[0089] 72h: The radioactivity of the lateral ventricles was 47.83 kBq / ml, and the radioactivity of the liver was 1.81 kBq / ml.

[0090] 96h: The radioactivity of the lateral ventricles was 34.50 kBq / ml, and the radioactivity of the liver was 1.75 kBq / ml.

[0091] 120h: The radioactivity of the lateral ventricles was 19.32 kBq / ml, and the radioactivity of the liver was 1.63 kBq / ml.

[0092] Control group: A weak signal appeared in the lateral ventricle at 0h after injection (radioactivity concentration 25.3kBq / mL), the signal spread to the whole brain at 1h, significant radioactive uptake was observed in the liver at 4h (18.7kBq / mL), and the signal in the lateral ventricle basically disappeared at 24h (<1kBq / mL).

[0093] Experimental group: Significant signal in the lateral ventricle 1 hour after injection (357.95 kBq / mL), diffused to the entire lateral ventricle and spinal aqueduct at 12 hours, and maintained a high signal in the lateral ventricle at 24 hours (96.81 kBq / mL). Liver uptake reached its peak at 48 hours (2.63 kBq / mL), and detectable signal was still present in the lateral ventricle after 120 hours (19.32 kBq / mL), indicating that the tracer imaging agent has a long retention time in the brain.

[0094] in conclusion: 89 Zr-DFO-HRP tracer imaging agent can achieve effective imaging in the lateral ventricle 1 hour after injection, with 83% of the drug concentrated at the direct injection point; the drug diffuses to the entire lateral ventricle and spinal aqueduct within 12 hours; liver metabolic signal reaches its peak at 2 days; even after 5 days (120 hours), the drug remains in the lateral ventricle, and the uptake by the liver and surrounding soft tissues is significantly reduced, extending the effective tracking window to 120 hours.

[0095] In its specific implementation, this invention employs an innovative bifunctional chelating agent: DFO (deferoxamine) is used as a bifunctional chelating agent, which can simultaneously achieve efficient coupling with HRP (horseradish peroxidase) and with... 89 The stable chelation of Zr (zirconium-89) solves the problems of low labeling efficiency, harsh reaction conditions, and weak affinity of traditional chelating agents (such as DOTA).

[0096] pH-sensitive labeling system: Establish a precise pH control system, adjusting the pH to 9.0 during the DFO and HRP coupling stage. 89 During the Zr labeling stage, the pH was adjusted to 7.0 to ensure efficient execution of each reaction step while avoiding the impact of extreme pH on protein activity.

[0097] Neuron-specific uptake optimization: By optimizing the preparation process of the tracer (such as the molar ratio of DFO to HRP, reaction temperature and time), the specific uptake ability of the tracer in lystic neurons is improved, enhancing the efficiency of non-synaptic transmission and laying the foundation for long-range, monosynaptic tracer.

[0098] In practical implementation, this invention has the following advantages:

[0099] 1. Design of bifunctional chelating agent: DFO (deferroamine) is selected as a bifunctional chelating agent. The amino group at one end can be specifically coupled to the carboxyl group (C-terminus) of HRP through an amide bond, and the hydroxamic acid group at the other end can form a stable six-membered ring chelate with 89Zr. This solves the problems of low labeling efficiency, poor stability and damage to protein activity of traditional chelating agents (such as DOTA). Moreover, DFO has a small molecular weight (about 560 Da) and low steric hindrance, which does not affect the transsynaptic activity and neuronal uptake capacity of HRP.

[0100] 2. pH-sensitive labeling system: A staged pH precision control strategy was established—DFO and HRP coupling stage (pH=9.0), an alkaline environment was maintained using Na2CO3 / NaHCO3 buffer to promote amide bond formation; 89Zr labeling stage (pH=7.0), the pH was adjusted to neutral using HEPES buffer to avoid the destruction of HRP activity by extreme pH, while ensuring that the chelation reaction between 89Zr and DFO proceeds efficiently (reaction efficiency ≥95%).

[0101] 3. Neuronal targeting optimization: By controlling the molar ratio of DFO to HRP (9:1), reaction temperature (37℃), and time (120min), the activity retention rate of HRP is maximized (≥90%). Combined with intraventricular injection, the tracer is directly introduced into the central nervous system, reducing non-specific uptake by peripheral tissues (such as liver and kidneys) and improving the specific uptake efficiency of hygroscopic neurons (10-15 times higher than intravenous injection).

[0102] The 89Zr-DFO-HRP contact neuron-specific tracer and imaging agent of this invention, through innovative design of chelating agent selection, labeling system construction, and optimized preparation process, specifically addresses the core pain points of traditional neuron tracing technologies (fluorescent labeling, traditional radioactive labeling, and simple HRP tracing). It exhibits significant advantages in four dimensions: in vivo imaging performance, labeling stability, operational practicality, and application scalability, as detailed below:

[0103] I. Live imaging performance: Breakthrough the technical bottlenecks of "short time, low resolution, and shallow penetration".

[0104] 1. Long-term tracking capability, covering the entire cycle of multi-level synaptic transmission in neurons:

[0105] Traditional fluorescently labeled tracers have an effective tracking window of only a few hours due to problems such as fluorescence bleaching and enzyme degradation; traditional radiolabels (such as 18F and 99mTc) are limited by the short half-life of the radionuclides (18F half-life is 110 minutes, and 99mTc half-life is 6 hours), which cannot meet the needs of dynamic observation of the physiological and pathological functions of neurons across synapses.

[0106] This invention utilizes 89Zr (half-life 78.4 hours), combining the highly stable chelation properties of DFO with 89Zr (stability constant lgK≈35), extending the effective tracking window of the tracer in vivo to 120 hours (5 days). Experimental data show that after lateral ventricle injection of 370 kBq tracer, the lateral ventricle still maintains a detectable radioactivity of 19.32 kBq / ml after 120 hours. This allows for the complete capture of the full-cycle dynamics of neurons from "initial uptake - physiological - pathological - long-term effects," providing sufficient temporal data for the study of long-range neural circuits (such as the cortex-thalamus-hippocampus circuit) between hygroscopic neurons and other functional nuclei in the brain.

[0107] 2. High spatial resolution accurately presents the fine structure and synaptic connections of tactile neurons:

[0108] Traditional FDG-PET imaging is affected by the non-specific uptake of tracer imaging agents, and the spatial resolution is usually only 3-5 mm, making it impossible to distinguish individual neurons or synaptic structures; fluorescent labeling is difficult to image deep brain tissues (such as the basal ganglia and spinal cord) clearly due to insufficient tissue penetration depth (≤500 μm).

[0109] This invention enhances resolution through two major design features: first, the neuron-specific transsynaptic properties of HRP target only neurons and synaptic structures, reducing non-specific uptake by glial cells, blood vessels, etc.; second, the positron emission properties of 89Zr (positron energy 0.903MeV), when matched with PET / MRI dual-modal imaging equipment, improve spatial resolution by 5-8 times compared to traditional FDG-PET (up to 0.5-1mm), clearly presenting neuronal neurite branches, presynaptic membrane distribution, and even capturing the "dynamic migration of tracer during synaptic transmission," providing a precise structural basis for analyzing synaptic connection patterns.

[0110] 3. Deep tissue penetration capability, enabling imaging of the entire brain and spinal cord neural pathways:

[0111] Fluorescent labeling relies on optical imaging, which is affected by the scattering and absorption of light by biological tissues (such as brain tissue). Its penetration depth is limited to the surface tissues (such as the superficial layer of the cerebral cortex) and cannot reach deep structures such as subcortical nuclei, brainstem, and spinal cord. Traditional radiolabeling (such as 99mTc) has low nuclide energy, which makes the signal of deep tissues easily attenuated, resulting in poor imaging signal-to-noise ratio.

[0112] This invention employs PET / MRI dual-modal imaging technology. The positrons emitted by 89Zr can penetrate several centimeters of biological tissue. Combined with lateral ventricle injection (the tracer imaging agent directly enters the central nervous system, avoiding the blood-brain barrier), it enables complete imaging of neural pathways throughout the whole brain and spinal cord. In experiments, the tracer imaging agent diffused throughout the entire lateral ventricle and spinal aqueduct within 12 hours after injection, clearly revealing the connections between the spinal cord and brain neural circuits, overcoming the limitation of traditional techniques that "can only observe superficial neurons."

[0113] II. Labeling stability: Solve the problems of "easy label removal and low activity" and ensure the reliability of experiments.

[0114] 1. High stability both in vitro and in vivo, avoiding interference from free nuclides:

[0115] Traditional chelating agents (such as DOTA) require high temperature (40-50℃) and long time (30-60 minutes) to bind with 89Zr, which can easily lead to denaturation of the coupled protein and easy delabeling after labeling (the radiochemical purity is usually less than 80% after 48 hours). Free radionuclides (such as 89Zr) can be non-specifically taken up by tissues such as liver and bone, interfering with imaging results.

[0116] This invention selects DFO as a bifunctional chelating agent and achieves two major stability advantages by optimizing the labeling conditions (reaction at 37°C for 15 minutes, neutral system at pH 7.0):

[0117] In vitro stability: Radiochemical thin-layer chromatography (R-TLC) showed that the tracer in human serum was 91.5±0.56% radiochemically pure at 24h, 89.5±0.63% at 48h, and 80.5±1.24% at 120h, which is far superior to the traditional DOTA labeling system (which is usually <85% radiochemically pure at 48h).

[0118] In vivo stability: The liver radioactivity was only 2.63 kBq / ml 48 hours after injection, and continued to decrease to 1.63 kBq / ml after 120 hours, indicating that there was very little free 89Zr, which avoided interference from metabolic organs on the imaging signal and ensured the accuracy of the data.

[0119] 2. Fully preserves HRP transsynaptic activity, ensuring targeted tracking:

[0120] Simple HRP tracing requires fixation in vitro and substrate color development, making it unsuitable for in vivo application; traditional radiolabeling (such as DOTA-labeled HRP) is prone to damage to the spatial structure of HRP and loss of transsynaptic activity due to harsh reaction conditions (high temperature, extreme pH) (activity retention rate <60%).

[0121] This invention achieves a HRP activity retention rate of ≥90% through staged pH control (pH = 9.0 during the DFO-HRP coupling stage and pH = 7.0 during the 89Zr labeling stage) and mild reaction parameters (37℃, 15-120 minutes reaction time). Experimental verification shows that the tracer imaging agent can enter the cell via neuronal endocytosis, penetrate the synaptic cleft, and enter the next level of neurons, achieving continuous tracking of synapses at levels 3 and above, thus solving the core problem of "protein inactivation after labeling".

[0122] III. Practicality of Operation: The process is simplified and the cost is reduced, making it suitable for widespread promotion.

[0123] 1. The labeling reaction conditions are mild and require no complex equipment:

[0124] Traditional DOTA labeling of 89Zr requires high temperature (40-50℃), strict pH control (5.5-6.5), and the addition of a catalyst (such as EDTA). The process is cumbersome (requiring over 60 minutes) and demands sophisticated equipment (a constant-temperature shaker and a precise pH control system are required). In contrast, the labeling process of this invention offers three advantages:

[0125] Simplified temperature control: The entire reaction process is carried out at 37°C (which can be achieved through a regular constant temperature water bath or nitrogen blowing device, without the need for high-temperature equipment);

[0126] Time simplification: The labeling reaction of 89Zr with DFO-HRP takes only 15 minutes, which is 75% shorter than DOTA labeling (30-60 minutes);

[0127] Simplified procedure: No catalyst is required; the reaction can be completed simply by adjusting the pH with Na2CO3 solution. Routine laboratory personnel can operate the procedure after minimal training.

[0128] 2. The purification process is highly efficient and the product recovery rate is high.

[0129] Traditional purification of radiolabeled products often requires high-performance liquid chromatography (HPLC), which is costly (hundreds of thousands of yuan), complex (requiring gradient elution and fraction collection), and has low product recovery (typically <70%). This invention employs a PD-10 desalting column + 100×10 3 Two-step purification method using ultrafiltration tubes:

[0130] The PD-10 desalting column can quickly remove free DFO and small molecule impurities (elution time <30 minutes);

[0131] Ultrafiltration tubes can concentrate products and remove free 89Zr, with a final product recovery rate of ≥90%, and the equipment cost is low (PD-10 column unit price is tens of yuan, ultrafiltration tubes can be reused), making them suitable for laboratory-scale preparation.

[0132] 3. Raw materials are readily available and costs are controllable:

[0133] The core raw materials used in this invention (DFO, HRP, and 89Zr) are all commercially available mass-produced products: DFO (deferoxamine) is a commonly used chelating agent in clinical practice, with a market price of approximately RMB 100 / 100mg; HRP (horseradish peroxidase) is a routine reagent in biological experiments, with a unit price of approximately RMB 50 / 100mg for products with a purity of over 95%; 89Zr can be routinely prepared using a medical cyclotron accelerator without special customization, significantly reducing R&D and application costs and facilitating promotion to research institutions and hospital research departments.

[0134] IV. Application Expansion: It covers multiple scenarios of neuroscience research and has broad potential.

[0135] 1. Compatible with PET / MRI dual-modal imaging to meet different research needs:

[0136] Traditional imaging agents typically support only a single imaging mode (fluorescent labeling only supports optical imaging, and 18F labeling only supports PET imaging), failing to combine the advantages of different imaging technologies; the tracer imaging agent of this invention can be adapted to both PET imaging (focusing on dynamic metabolic tracking) and MRI imaging (focusing on anatomical structure localization).

[0137] PET mode: It can quantify the changes in the radioactivity of tracer in different brain regions and dynamically analyze the rate of neuronal uptake and transsynaptic transmission.

[0138] MRI mode: It can precisely locate the anatomical structures of brain regions where the tracer is located (such as the CA1 region of the hippocampus and the ventral posterior nucleus of the thalamus), and realize the dual analysis of "functional dynamics + structural localization", providing more comprehensive data support for neural circuit analysis and neuronal function research.

[0139] 2. Applicable to a variety of animal models, seamlessly connecting basic research to preclinical studies:

[0140] Traditional fluorescently labeled imaging agents are limited in penetration depth and are only suitable for small / ex vivo models such as mouse brain slices and zebrafish. The tracer imaging agent of this invention has strong deep penetration ability and long-term tracking stability, and can cover a variety of animal models from mice and rats to non-human primates (such as rhesus monkeys).

[0141] Small animals (mice / rats): can be used for basic neuroscience research (such as synaptic plasticity, circuit formation during neural development);

[0142] Non-human primates: can be used for preclinical research (such as observation of neural circuit damage in Parkinson's disease and Alzheimer's disease models), laying the foundation for subsequent clinical translation (such as early diagnostic research of neurodegenerative diseases), with far more application scenarios than traditional tracing technology.

[0143] 3. No tissue damage, ensuring the authenticity of the experimental subjects' physiological state:

[0144] Traditional fluorescent labeling requires electroporation or microinjection to damage the neuronal membrane to introduce the marker, which may alter the normal physiological functions of neurons (such as electrical activity and neurotransmitter release). The tracer imaging agent of this invention enters the central nervous system through lateral ventricle injection (a minimally invasive procedure requiring only a 10 μL microsyringe). HRP can be taken up by the neurons through natural endocytosis without damaging the cell membrane. This allows for a true reflection of the transsynaptic transmission patterns of neurons in their "physiological state," avoiding experimental bias caused by procedural damage and improving the reliability of research data.

[0145] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown throughout are only one of the embodiments of the present invention. The actual structure is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A specific tracer and imaging agent for touch-sensitive neurons, characterized in that, This includes horseradish peroxidase, zirconium-89 isotopes chelated by deferoxamine, and pharmaceutically acceptable carriers.

2. The tactile neuron-specific tracer imaging agent according to claim 1, characterized in that: The deferoxamine and horseradish peroxidase achieve specific binding through C-terminal modification.

3. The tactile neuron-specific tracer imaging agent according to claim 1, characterized in that: The specific activity of the tracer imaging agent ranges from 18.5 to 74 MBq / mg.

4. The ionotropic neuron-specific tracer imaging agent according to claim 1, characterized in that: The pharmaceutically acceptable carrier is selected from at least one of HEPES buffer, Na2CO3 / NaHCO3 buffer, and acetate / sodium acetate buffer.

5. The tactile neuron-specific tracer imaging agent according to claim 1, characterized in that: The radiochemical purity of the tracer in serum was determined by radiometric thin-layer chromatography as follows: 91.5±0.56% at 24 hours, 89.5±0.63% at 48 hours, and 80.5±1.24% at 120 hours.

6. A method for preparing a hygroscopic neuron-specific tracer imaging agent as described in any one of claims 1-5, characterized in that: Includes the following steps: S1: Preparation of DFO-HRP solution: (1) Dissolve ferricamine in DMSO to prepare a DFO solution with a concentration of 20 μg / μl; dissolve horseradish peroxidase in ultrapure water to prepare an HRP solution with a concentration of 10 μg / μl; (2) Take HRP solution and add 0.1 mol / L Na2CO3 / NaHCO3 buffer solution, pH=9.0, and add 0.1 mol / L Na2CO3 solution dropwise to adjust the pH of the system to 9.0; (3) According to the molar ratio of DFO to HRP of 9:1, DFO solution was added to the above HRP solution in 3 portions, with an interval of 10 min between each portion, and the reaction was carried out in a nitrogen blower at 37℃ for 120 min. (4) The reaction products were sequentially purified by PD-10 desalting column separation and 100×10 3 The ultrafiltration tube was concentrated and washed twice to collect the DFO-HRP solution. S2: 89 Preparation of Zr-DFO-HRP: (1) Take 89 Zr was added to 9 volumes of 0.1 mol / L HEPES buffer, and the pH of the system was adjusted to 7 by adding 1 mol / L Na2CO3 solution. 89 Zr solution; (2) 89 Zr solution was added to DFO-HRP solution and reacted at 37°C for 15 min to obtain a tracer imaging agent.

7. The use of a hygroscopic neuron-specific tracer as described in any one of claims 1-5 in in vivo transsynaptic imaging of neurons for non-therapeutic purposes.

8. The application according to claim 7, characterized in that: The in vivo imaging is a PET / MRI dual-modal imaging.

9. The application according to claim 7, characterized in that: The tracer imaging agent used in this application is administered via subarachnoid injection into the lateral ventricle or lumbar anesthesia site, enabling long-range dynamic tracing of multi-level neuronal circuits with an effective tracking window of 6-120 hours.

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