An MRI-CEST polypeptide probe, imaging method and device for targeted identification of abnormal α-synuclein in the brain of Parkinson's disease

Abstract

The present invention relates to an MRI-CEST polypeptide probe, imaging method and equipment for targeting and identifying abnormal α-synuclein in the brain of Parkinson's disease. The sequence of the polypeptide probe is: TFFYGGSRGKRNNNKTEEY. The implementation of the equipment includes the following steps: (1) using Fmoc solid phase to synthesize the targeted polypeptide probe; (2) the targeted polypeptide probe binds to abnormal α-synuclein in the brain, taking into account high-quality CEST signals; (3) the MRI-CEST sequence is used to collect brain images of Parkinson's rats, and the images before and after the injection of the polypeptide probe are collected respectively, and the abnormal α-synuclein deposition in the model brain is evaluated in a non-invasive and radiation-free method; (4) the collected images are post-processed by Matlab, and the protein deposition is semi-quantitatively evaluated. The present invention is expected to improve the accuracy of early diagnosis of PD.

Description

Technical Field

[0001] The present invention relates to the field of medical imaging technology, and specifically designs a polypeptide probe targeting abnormal alpha-synuclein in the brain of Parkinson's disease, and an imaging method and device thereof. Background Art

[0002] Parkinson's disease (PD), also known as parkinsonism, is the second most common degenerative disease of the central nervous system after Alzheimer's disease. The population base of people suffering from Parkinson's disease in my country is huge, which will impose a heavy burden on public health, family, society and economy, and have a serious impact on the care and services for the elderly in a rapidly aging society. At present, the diagnosis of PD is mainly based on its typical clinical manifestations, namely motor symptoms such as bradykinesia, muscle rigidity and resting tremor. However, at this time, 50-80% of the dopaminergic neurons in the brain have died, and it is already in the middle and late stages of the disease. PD patients are currently incurable, and early accurate diagnosis and timely treatment intervention are the best ways to improve patient prognosis.

[0003] Due to spontaneous or external factors, α-synuclein in the brain of PD patients misfolds and abnormally aggregates to form transneuronal inclusions (i.e., Lewy bodies and Lewy neurites), which is the main pathological hallmark of PD. Although the pathogenic mechanism of abnormal α-synuclein is still unclear, studies have shown that α-synuclein is already present in the brain of PD patients in the early stages of the disease, and its distribution is related to clinical symptoms and disease progression. Therefore, if abnormal aggregated α-synuclein in the brain can be detected, it will be expected to improve the accuracy of early clinical diagnosis of PD patients and buy more time for treatment intervention for patients.

[0004] MRI has unique advantages in in vivo imaging. Its imaging of anatomical structures has become close to perfect with the increase of magnetic field strength and the acquisition of multi-channel signals. Chemical exchange saturation transfer (CEST) imaging is a new magnetic resonance molecular imaging technology developed based on magnetization transfer and chemical exchange theory. It is currently a research hotspot for new imaging methods at home and abroad. Although MRI-CEST technology has developed rapidly, there are very few studies involving CEST technology to detect α-synuclein. How to use MRI-CEST technology to accurately quantify α-synuclein in the brain of PD patients is still challenging.

[0005] Angiopep-2 is a small peptide composed of 19 amino acids derived from the Kunitz domain of aprotinin, with an amino acid sequence of TFFYGGSRGKRNNFKTEEY, which can trigger transcellular action and cross the blood-brain barrier (BBB) ​​by recognizing LRP-1 expressed by brain capillary endothelial cells. Compared with other targeting ligands.

[0006] Previous studies have shown that Angiopep-2 has a strong CEST effect at 3.2 ppm, which confirms that Angiopep-2 has a CEST effect and is concentration-dependent, making it a good MRI-CEST contrast agent. However, studies have shown that Angiopep-2 has an affinity for both the Aβ protein of Alzheimer's disease and the α-synuclein of PD, and lacks specificity. In view of this, improving the specificity of Angiopep-2 is a good strategy to achieve targeted diagnosis of abnormal α-synuclein in the brain of Parkinson's disease. Summary of the invention

[0007] In order to overcome the above problems, the present invention provides a polypeptide probe that can target abnormal α-synuclein in the brain of Parkinson's disease. The structure can significantly reduce the affinity for Aβ protein, thereby improving the specificity for α-synuclein. The present invention also provides an imaging method and system designed in combination with the probe.

[0008] The present invention provides a polypeptide probe that can target abnormal α-synuclein in the brain of Parkinson's disease, and its amino acid sequence is: TFFYGGSRGKRNNNKTEEY. It is a derivative of Angiopep-2, formed by mutation of the 14th phenylalanine of Angiopep-2 to asparagine, and its structural formula is as follows:

[0009]

[0010] The present invention adopts the following technical solutions:

[0011] 1. Preparation of targeted peptide probes:

[0012] The peptides were synthesized using the Fmoc solid phase peptide synthesis strategy. This strategy uses Fmoc-Asn(pbf)-OH as a raw material to bond with Wang resin, and then other amino acids are added in sequence. After the synthesis is completed, the cleavage reagents TFA, water, and TIS are added for cleavage reaction, and finally the precipitate is collected and purified using a C18 reverse phase preparative column.

[0013] 2. A method for imaging a targeted peptide probe in a Parkinson's disease model, comprising the following steps:

[0014] 2.1 Scan the MRI-CEST image of the brain of the PD rat model before injection of specific peptides as a template, use the Scout sequence for positioning, scan the T2 image and select the target level with the maximum field of view of the striatum on the coronal section for further CEST imaging;

[0015] 2.2 Inject specific peptides and collect brain MRI-CEST images again at 30min, 60min and 90min, with the scanning parameters and layers consistent with the mold;

[0016] 2.3 The collected images were post-processed using Matlab. The image post-processing was centered on the saturation frequency of water, and a "negative-positive" symmetrical subtraction process was performed on the 121 saturated images and 1 unsaturated image collected by the CEST sequence to obtain a CEST pseudo-color image to compare the changes in CEST signal intensity before and after injection, so as to achieve the localization and semi-quantitative detection of abnormal α-synuclein in the brain.

[0017] 3. The present invention provides an imaging device for a Parkinson's disease model using a polypeptide probe, including a chemical exchange saturation transfer (CEST) imaging system, and also includes a program module, wherein the program module sets a method for imaging a targeted polypeptide probe in a Parkinson's disease model. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below with reference to the embodiments.

[0019] 1. Preparation of targeted peptide probes:

[0020] The Fmoc solid phase peptide synthesis strategy is adopted, and the general structural formula of the targeted probe is shown in the sequence table. This strategy uses Fmoc-Asn(pbf)-OH as a raw material to bond with Wang resin. Then, after removing the Fmoc group, a condensation reaction is carried out with Fmoc-Typ(tBu)-OH in the presence of condensation agents DIC and HOBt to complete the connection of the second amino acid; then, Fmoc is removed and a condensation reaction is carried out with the third amino acid Fmoc-Val(Boc)-OH; according to this procedure, the amino acids used include Fmoc-Gly-OH, Fmoc-Arg-OH and Fmoc-Trp-OH, and the synthesis is completed from the C-terminus to the N-terminus, until the synthesis is completed, and a resin with side chain protection removed from the Fmoc group is obtained. A cleavage agent was added to the peptide resin obtained above, wherein the cleavage agent was mixed with TFA, water and TIS (triisopropylsilyl chloride) in a mass ratio of 96:1.5:2.5, reacted at 20°C in the dark for 2 hours, filtered; the filtrate was collected, concentrated by rotary evaporator, and then about 10 times the volume of pre-cooled anhydrous ether was added, precipitated at -20°C for 3 hours, a white powder was precipitated, the precipitate was collected, and then the precipitate was washed with anhydrous ether and vacuum dried to obtain a polypeptide. Among them, a C18 reverse phase preparative column was used for purification, the main peak was collected, and freeze-dried to obtain a white solid as the product. The product was identified by mass spectrometry, and the molecular weight shown in the mass spectrum was basically consistent with the theoretical molecular weight.

[0021] 2. A method and system for imaging a targeted peptide probe in a Parkinson's disease model, comprising the following steps:

[0022] 2.1 Scanning MRI-CEST images of the brain of PD rat models before injection of specific peptides were used as templates. PD rat modeling: unilateral striatal injection of α-synuclein preformed precursor fibers (PFFs) was used. SD rats were anesthetized with 3mg / ml sodium pentobarbital. They were then fixed in a stereotaxic instrument, the surface hair was removed, the scalp was exposed, and the skin and surface fascia were cut with straight scissors. The origin of the stereotaxic instrument was fixed at the rat's anterior bregma, and then the coordinates were moved to the injection site (striatum AP+1.6, ML+2.4, DV-4.2). A dental drill was used to drill a hole, and a Hamilton microsyringe (specification 10ul) was used to inject α-synuclein PFFs (4ul / 8ug) at a rate of 1ul / min. Subsequently, the wound was sealed with sterile wax as an additional anti-inflammatory measure. The rats were kept for 4 months after surgery for further scanning.

[0023] Brain MRI-CEST image scanning: First, the rat brain was at the target level of T2WI. The Scout sequence was used for positioning to adjust and determine the position of the rat brain so that the brain was located in the center of the coil. The 7TMRI T2WI parameters were as follows: FOV = 45mm × 45mm, TR = 2000ms, TE = 4.1ms; Averages = 2, data matrix = 128 × 128, slice thickness = 2.0mm, and slice spacing = 0.01mm. The maximum field of view of the striatum was used as the target level on the coronal section. Since the CEST sequence is sensitive to magnetic field inhomogeneity, field shimming was performed before scanning the CEST image. The field shimming condition was FWHM less than 15HZ, and continuous wave was used for pre-saturation. The continuous wave saturation pulse sequence of echo sequence imaging was used to acquire brain CEST images, and the chemical shift range was -6.0ppm to +6.0ppm.

[0024] 2.2 Injection of specific peptides, which are solid peptides dissolved in PBS, with a concentration of 30 mM. Brain MRI-CEST images were collected at 30 min, 60 min and 90 min after injection, and the scanning parameters and layers were consistent with the mold.

[0025] 2.3 After the experiment, the collected images were post-processed by Matlab. The image post-processing was centered on the saturation frequency of water, and a "negative-positive" symmetrical subtraction process was performed on the 121 saturated images and 1 unsaturated image collected by the CEST sequence to obtain a CEST pseudo-color image to compare the changes in CEST signal intensity before and after injection, so as to achieve the localization and semi-quantitative detection of abnormal α-synuclein in the brain.

[0026] The protection scope of the present invention is limited only by the claims. With the help of the teachings of the present invention, those skilled in the art will easily recognize that alternative structures of the structures disclosed in the present invention can be used as feasible alternative embodiments, and the embodiments disclosed in the present invention can be combined to generate new embodiments, which also fall within the scope of the appended claims.

Claims

1. An MRI-CEST polypeptide probe for targeting and identifying abnormal α-synuclein in the brain of Parkinson's disease, characterized in that: Its amino acid sequence is: TFFYGGSRGKRNNNKTEEY.

2. An imaging method using the specific polypeptide probe according to claim 1 in a Parkinson's disease model, characterized in that: include: (1) Scanning the MRI-CEST images of the brain of PD rat models before injection of peptide probes as templates; (2) inject the peptide probe and perform brain MRI-CEST imaging again at different time points; (3) The acquired images were post-processed using Matlab and the changes in CEST signal intensity before and after injection were compared to achieve localization and semi-quantitative detection of abnormal α-synuclein in the brain.

3. The imaging method according to claim 2, characterized in that: Step (1) The Scout sequence was used for positioning, the T2WI images were scanned, and the target layer with the largest field of view of the striatum on the coronal section was selected for further CEST imaging.

4. The imaging method according to claim 2, characterized in that: The acquisition time points in step (2) are 30 min, 60 min and 90 min, and the scanning parameters and layers are consistent with the template.

5. The imaging method according to claim 2, characterized in that: Step (3) Image post-processing: With the saturation frequency of water as the center, 121 saturated images and 1 unsaturated image acquired by the CEST sequence are subjected to "negative-positive" symmetric subtraction processing to obtain a CEST pseudo-color image; the asymmetric transfer rate value of each voxel constituting the image is calculated and its mean value is statistically calculated to quantify the CEST signal intensity.

Images