| BackgroundAlzheimer’s disease (AD), the leading cause of dementia in the elderly, is an irreversible, progressive neurodegenerative disorder pathologically characterized by extracellular accumulation of amyloid-β(Aβ) senile plaques (SPs), intracellular neurofibrillary tangles (NFTs), and loss of neurons and synapses, clinically characterized by memory loss and cognitive decline, leading invariably to death, usually within 7-10 years after diagnosis. AD not only has devastating effects on the sufferers and their caregivers, but it also has a tremendous socioeconomic impact on families and the health system; a burden which will only increase in the upcoming years as the population of most countries ages. Symptomatic treatment with an acetylcholinesterase inhibitor or a glutamatergic moderator provides modest benefit in some patients usually by temporary stabilization rather than a noticeable improvement in memory function. There is no cure for AD nor proven way to slow the rate of neurodegeneration. AD constitutes one of the major health problems in the world because no cure is found.In the absence of biologic markers, direct pathologic examination of brain tissue derived from either biopsy or autopsy remains the only definitive method for establishing a diagnosis of AD. The current diagnosis methods include clinical tests, magnetic resonance imaging (MRI), positron emission tomography (PET), and cerebrospinal fluid (CSF) biomarkers. Presently, diagnosis of AD is made according to the NIH-ADAD criteria by the application of several neuropsychological tests that exclude other potential causes of dementia. The degree of accuracy for this method ranges from 50 to 90%. As such, clinical diagnosis of AD can only be truly confirmed histopathologically, by the observation of a large amount of NFTs and neuritic plaques in the neocortex of postmortem brain tissue.Thus, it is evident that scientific efforts in translating neuroscience knowledge into reliable and non-invasive diagnosis tools are highly valuable. A validated biomarker in brain would likely prove useful to identify and follow individuals at risk for AD and to assist in the evaluation of new therapies currently under development. Some researches devote themselves to develop radiolabeled specific imaging agents with high brain entry and high levels of specific binding in brain regions known to contain high concentrations of Aβ. PET is a sensitive molecular imaging technique that allows in vivo quantification of radiotracer concentrations in the picomolar range, allowing the non-invasive assessment of molecular processes at their sites of action, detecting disease processes at asymptomatic stages when there is no evidence of anatomic changes on computerized tomography (CT) and MRI. Autopsy brain studies cannot directly address the issue of progression of amyloid deposition in an individual over time. Imaging by means of PET is therefore a valuable tool with which to follow the natural progression of the disease. What’s more, PET is an imaging technique that can be used to monitor drug pharmacokinetics non-invasively in patients, based on radiolabeling these drugs with short-lived positron emitters.The molecular imaging probes for the non-invasive in vivo quantification of SPs and NFTs burden in the brain has revolutionized the approach to the early diagnosis and evaluation new treatment of AD, because they allow the in vivo assessment of brain Aβ and hyperphosphorylated tau protein as well as their changes over time, providing highly accurate, reliable, and reproducible quantitative statements of regional or global SPs and NFTs burden in the brain, essential for therapeutic trial recruitment and for the evaluation of anti-A(3 and tau-hyperphosphorylated treatments. It can be concluded that the use of PET in the evaluation of AD has alleviated the early diagnosis of the disease considerably. The development of new tracers with even better properties and tracers labeling amyloid fibrils and neurofibrillary tangles is therefore still a highly prioritized research area.As a quantitative neuroimaging probe, specific radiotracers have to possess a number of key general properties:they should be lipophilic allowing crossing the blood brain barrier, preferably not be metabolized, while reversibly binding to Aβ in a specific and selective fashion. An important part of the development of new potential tracers for visualizing amyloid with PET is the preclinical evaluation of these compounds.ContentsPart I Improved preparation and optimization of [18F]-THK523Objective:Although for many years "Aβ-centric" hypotheses dominated AD research, the importance of tau in the pathogenesis of AD is now much more appreciated. It was found that increased tangle load, but not cell loss, atrophy, neuritic plaque load in the hippocampal neuropathological variables, was associated with increased severity of aggressive behaviours and presence of chronic aggression. What’s more, studies have confirmed that, during the course of AD, NFTs in the hippocampus and enthorinal cortex are positively correlated with the cognitive decline. "Paired helical filaments" (PHFs) formed by hyperphosphorylated forms of the cytoskeletal protein tau are the major components of NFTs. To improve and optimize the radiosynthesis of [18F]-THK523, We synthesized new precursor, establish fully automated synthesis and quality control standard of [18F]-THK523.Methods:we report a new protected precursor, 2-(2-(4-(tert-butoxycarbonyl)phenyl)quinolin-6-yloxy) ethyl 4-methylbenzenesulfonate (THK-7), instead of 2-((2-(4-aminophenyl)quinolin-6-yl)oxy)ethyl 4-methylbenzenesulfonate (BF241), and an improved automated radiosynthesis of [18F]-THK523 and the study of the chemical kinetics of the labeling reaction of [18F]-THK523. In addition to RHPLC and TLC analysis of [18F]-THK523, we established physical, chemical and biological characteristics of [18F]-THK523 for its quality control.Results:Quality controls of precursor TIIK-7 and THKF-2 were carried out. The structure identification and molecular weight were conformed. The purity was more than 99%for THK-7 and THKF-2. The baterial endotoxins and sterility were tested and conformed. Radio-High Performance Liquid Chromotography (radio-HPLC) analysis showed the radiochemical purity (RCP) of [18F]-THK523 was≥90%. The retention time (5R) of [18F]-THK523 and precursor THK-7 were 6.12min and 12.94min, respectively. The chemical identity of [18F]-THK523 was verified by co-injection with the non-labelled standard THKF-2 (tR=5.93 min). [18F]-THK523 for injection was clear and transparent, pH 6.6-7.0, radioactive half-life was 109+2 min, radioactive concentration was 12.3 mCi/mL, ethanol content was<10%, permeation pressure of filter membrane was≥0.4 MPa, bacteriology and endotoxin tests were negative. [18F]-THK523 has been achieved with high-yield (70±5%, n=6, decay-corrected to end of bombardment) as well as high radiochemical purity (>90%) and specific activity (2.5 ± 0.5 Ci/μmol) from protected precursor on fully automated module at the end of radiosynthesis (45-55 min). Mean labeling yields and mean specific radioactivity increased with reaction temperature and time. For reaction chemical kinetics parameters, rate constant (k) increases with the increase of temperature, indicating that high temperature is conducive to increased reaction rate. K didn’t change greatly with the concentration of THK-7. The chemical kinetics for [18F]-THK523 demonstrated that nucleophilic substitution could be carried out easily with protected precursor.Part Ⅱ Biological characteristics studies of [18F]-THK523 for Tau imagingObjective:Metabolism and biodistribution patterns need to be evaluated for any candidate radiopharmaceutical that is being considered for clinical translation. To investigate of the biological characteristics of [18F]-THK523 and to evaluate whether it is an excellent brain tracer.Methods:Stability assessment of the complex was carried out by measuring its radio chemical purity at several time pints at 25 ℃ within 4 h. Lipid-water partition coefficient of [18F]-THK523 was measured phosphate-buffered saline (PBS) (pH= 7.4) and n-octyl alcohol. The charge of [18F]-THK523 was determined by paper electrophoresis using kalium phosphate buffer solution:alcohol:distilled water. Plasma protein binding rate was measured by four dosages. The blood clearance patterns of [18F]-THK523 were simulated using Pharmacokinetics Local Model (PLM) software. Micro PET imaging and biodistribution of [18F]-THK523 in mice were carried out. Autoradiography is a commonly used method in the study of radioligands, as direct comparisons of in vitro autoradiography images can be made with in vivo PET images. The kinetics needs to be suitable for PET, i.e. the binding equilibrium should be reached in the time frame of a PET experiment. These and other important criteria for a suitable PET ligand need to be studied in the preclinical evaluation phase. Acute toxicity test was evaluated with different dosage of [18F]-THK523.Results:[18F]-THK523 was radiosynthesized on automated module and biological characteristics of [FJ-THK523 were evaluated. In vitro studies demonstrated that [18F]-THK523 was electrically neutral, lipophilic (lgP= 0.99±0.06, n= 7) and quite stable with its radiochemical purity of more than 90% maintained for up to 4 h at room temperature. Due to its low molecular weight, [18F]-THK523 can easily cross blood brain barrier (BBB). With relatively low plasma protein binding rate, [18F]-THK523 didn’t show mass concentration-dependent characteristics. Pharmacokinetics parameters of the blood were calculated, blood kinetic studies revealed the dual-exponential equation was Y= 2.23e-0.014t+1.89-000071 with parameters of t1/2α 47.9247 min-1, t1/2p 965.1007 min-1, K120.0067 min-1, K21 0.0070 min-1, Ke 0.0015 min-1, Plasma Clearance (CL) 0.0359 ID%g-1 min-1, area under concentration-time curve (AUC) 2785.08 ID%g-’min. The radiopharmaceutical potential of [18F]-THK523 in animals was investigated and it was found that [18F]-THK523 had high brain uptake. The biodistribution of [18F]-THK523 was studied in healthy C57 mice. [18F]-THK523 was mainly metabolized by liver and excreted through biliary. Combined previous studies, [18F]-THK523 might be a promising candidate for further molecular imaging of tau pathology. With low molecular weight, [18F]-THK523 was stable, electrically neutral, lipophilic and non-mass concentration-dependent. No significant response showed in acute toxicity test. Preliminary biological studies have shown the excellent properties of [18F]-THK523 as brain imaging tracer for further research.Part III Automatic synthesis of [11C]-PIB and its related imaging studiesObjective:To improve the automated radiosynthesis of [11C]-PIB, improve their radiochemical purity and specific activity, reduce clinical injection pain. PMOD software was applied to analyze [11C]-PIB PET/MRI imaging. All of these will provide effective quantitative analysis for [11C]-PIB PET brain analysis. The analysis methods will provide a reference for the clinical application of Tau protein imaging.Methods:TRACERlab FXc automation module was applied to radiosynthesize [11C]-PIB. [11C] CO2 was changed into 11CH4 with Ni catalyst under 400℃.11CH4 reacted with iodine at 730℃ to afford 1CH3I. 11CH3I reacted with Ag-Triflate at 190℃ to afford 11C-Triflate-CH3I. "C-Triflate-CH3I reacted with precursor 6-OH-BTA-0 to afford [11C] 6-OH-BTA-1, i.e., [11C]-PIB. The quality control of [11C]-PIB was analyzed after the semi-preparative purification.17 cases, who were clinically diagnosed as aMCI, underwent MRI and PET/CT examination. PMOD software was applied to analyze brain uptake of [11C]-PIB in different regions.Results:The specific activity, radioactive concentration of [11C]-PIB was greatly improved with the new module. All of these were conducive to clinical applications of [11C]-PIB. The quality control of [11C]-PIB was tested. [11C]-PIB was clear and transparent without precipitation, its pH was between 6.9 to 7.0, sterile filtration permeability pressure was more than 0.4 MPa, the ethanol content was less than 10%, RCP of [11C]-PIB was more than 95%, The radioactive concentration was 17.6 mCi/ mL, radioactive half-life was 20+2 min, the specific activity was 4±0.3 Ci/μmol, bacteriology endotoxin test was negative. All the indicators were conformed to "guiding principles of quality control for positron radiopharmaceuticals".17 cases were clinically diagnosed as aMCI according to comprehensive analysis of MMSE score, AVLT delayed recall, Boston Naming Test, Trail Making Test B, animal verbal fluency, Hachinski ischemic clinical dementia rating score. They underwent PET/MRI examination. Both quantitative and visual assessment of PiB-PET images present a pattern of PiB retention that seems to replicate the sequence of Aβ deposition found at autopsy, with initial deposition in orbitofrontal cortex, inferior temporal, and gyrus rectus, followed by the cingulate gyrus and precuneus, the remaining prefrontal cortex and lateral temporal cortex, and finally to the parietal cortex. According to PMOD analysis, there were [11C]-PIB depositions in frontal lobe, parietal lobe, temporal lobe, occipital lobe, precuneus, hippocampus, posterior cingulate cortex, brainstem, thalamus, white matter on 13 cases, especially in frontal lobe, parietal lobe, temporal lobe, occipital lobe, precuneus, posterior cingulated. However, there were no significant [11C]-PIB depositions in these regions on the rest 4 cases. PMOD provides a quantitative method for brain regions analysis. The positive rate of screening aMCI was 76.5% according to [11C]-PIB PET examination among 17 cases clinically diagnosed as aMCI. What’s more, further research is needed to follow up these cases. Clinical analysis methods of [11C]-PIB PET will provide a methodology reference for the clinical application of Tau protein imaging.Part IV The synthesis, radiolabelling and biological characteristics study of a novel PET imaging probe [18F]-TKP for targeting amyloid and tau proteinObjective:To develop a molecular imaging probe to assay and quantify AP and tau in vivo for the early diagnosis and efficacy evaluation of therapeutic treatment of Alzheimer’s disease (AD).Methods:THKP as reference standard and its mesyloxy derivative TKPF as precursor for labeling with fluorine-18 were synthesized and identified. The quality control of THKP and TKPF was carried out. [18F]-TKP was automatically synthesized via nucleophilic substitution using one pot and two-pot reaction on Tracerlab FX N Pro radiosynthesis module. Quality control of [18F]-TKP, such as labelling yields, synthesis time and the specific activity of the final product [18F]-TKP were compared between one pot and two-pot reaction. In vivo quantitative microPET studies in wild type SD rats and C57 mouse were conducted to evaluate the in vivo pharmacokinetic profile and binding specificity. PMOD software was applied to evaluate the radio-uptake of [18F]-TKP in brain in detail. Acute toxicity test was evaluated with different dosage of [18F]-TKP.Results:A validated biomarker of intracellular amyloid and NFTs deposition in brain would likely prove to be useful for identifying and following individuals at risk of AD and for assisting the evaluation of new anti-amyloid and anti-NFTs therapies currently under development. A fully automated synthesis of [18F]-TKP-along with the synthesis and characterization of non-radioactive TKPF are reported. THKP and TKPF were successfully synthesized and the structures were confirmed by NMR. and high resolution MS, the overall yield of THKP and TKPF were 80% and 70%, respectively. We completed the synthesis of the precursor and standard for [18F]-TKP and radiosynthesis on two different methods, optimizing overall total synthesis time, specific radioactivity, product purity and synthesis automation. Inorganic and water-soluble materials in the reaction mixture were first removed by sequential purification on silica Sep-Pak cartridges on two-pot radiosynthesis. In this way, the efficiency of purification on the reverse phase HPLC column was enhanced. With this method, production of large amounts of [18F]-TKP with high chemical and radiochemical purities ready for administration to animals or human subjects was guaranteed. The quality control of [18F]-TKP was conformed to the "quality control of positron radiopharmaceuticals guiding principles" issued by SFDA. The radiochemical purity of [18F]-TKP was greater than 98%after HPLC preparation. The structure of the [18F]-TKP was verified by HPLC through coinjection with reference standard THKP. After semipreparative HPLC, [18F]-TKP was isolated from the mobile phase via a solid-phase extraction proce-dure (tC18 Sep-Pak Vac cartridge). Pure [18F]-TKP was recovered from tC18 Sep-Pak with absolute EtOH. Because of the hydrophobic nature of [18F]-TKP, the trapping efficiency by the tC18 Sep-Pak Vac column exceeded 90%. The recovery efficiency of [F]-TKP from the tC18 Sep-Pak with EtOH also exceeded 90% with a volume as small as 1 mL, which is advantageous for preparing concentrated solution of [F]-TKP in 10% EtOH in normal saline. Such concentrated solutions are convenient for rodent micro PET imaging experiments as they contain a sufficient amount of activity (1-3mCi) in small volumes (250-500 mL) for safe intravenous administrations. A solution of [18F]-TKP for human PET scanning was prepared from the [18F]-TKP solution in 10% EtOH in normal saline by diluting it with an equal volume of 25% solution of human serum albumin (HSA), which ensures sufficient stability and solubility. This solution was sterilized by filtration through a sterile membrane (0.22mm) filter. This sterile filter showed little radioactivity retention (3-5% of applied [18F]-TKP activity). HPLC analyses have demonstrated that [18F]-TKP is stable in both EtOH/saline/HSA and the EtOH stock solutions in excess of 6 hours. [18F]-TKP was obtained in a range of 18± 3%(n=6, one pot reaction) and 25 ±5%(n=6, two-pot reaction) radiochemical yields. The total synthesis time of [18F]-TKP were 45-55 min and 55-70 min and the specific activities were 0.7 ± 0.2 Ci/νmol and 1.5 ± 0.2 Ci/μmol at the end of synthesis for one pot and two pot, respectively (decay corrected to end of bombardment, EOB). The PMOD analysis of PET/MRI imaging revealed that the highest brain uptake of [18F]-TKP was at 2 min after injection in most parts of brain, such as amygdala, caudate putamen, cortex (cortex auditory, cortex cingulate, cortex entorhinal, cortex frontal, cortex insular, cortex medial prefrontal, cortex motor, cortex orbito frontal, cortex somato sensory, cortex visual), hippocampus, hypothalamus, olfactory, midbrain, ventral tegmental area, ventral tegmental area, thalamus whole, pituitary and pons, et al. [18F]-TKP was then washed rapidly and then reach a plateau at 30min postinjection, then there is a slow increase. The highest regional uptake occurred at olfactory, followed by pituitary, amygdala, hypothalamus, pons, ventral tegmental area, midbrain, hippocampus, cortex, caudate putamen and thalamus whole. The olfactory cleaned most rapidly, whereas the hippocampus and cortex did relatively slow. For the cortex, the cortex entorhinal displayed the highest uptake and the lowest clearance, followed by cortex visual, cortex insular, cortex orbito frontal, cortex auditory, cortex cingulate, cortex frontal, cortex medial prefrontal, cortex motor and cortex somato sensory. The biodistribution revealed that [F]-TKP was primarly metabolized by liver. The acute toxicity of [18F]-TKP was negative. [18F]-TKP might be a useful PET radiotracer for targeting amyloid and tau protein.Part V Radiosynthesis optimization of [11C] labeled positron radiopharmaceuticals [11C]-TKF and its biological characteristics evaluationObjective:We designed a new probe [11C]-TKF targeted Tau protein on the basis of preliminary research. In addition, we tested different radiolabeled methods and established the automated radiosynthesis processes. The biological characteristics of [11C]-TKF was evaluated.Methods:The standard of [11C]-TKF, CTKF, was synthesized and identified by nuclear magnetic resonance. The quality control of [11C]-TKF was verified. The radiosynthesis of [11C]-TKF was optimized by comparing 11C-CH3I and 11C-Triflate-CH3I. The quality control of [11C]-TKF was tested. Biodistribution and toxicity of [11C]-TKF in normal C57 mice were evaluated.Results:The standard CTKF of [11C]-TKF was synthesized and identified. Quality control revealed that [11C]-TKF was sterile and baterial endotoxins was negative. The chemical purity of [11C]-TKF was more than 99%. [11C]-TKF was radiosynthesized by both 11C-CH3I and 11C-Triflate-CH3I. RHPLC demonstrated that there was the same retention time (5.5 min) for the coinjection of [11C]-TKF and reference standard CTKF. The total radiosynthesis time of [11C]-TKF was 35-40 min through 11C-Triflate-CH3I, the labeling yields and specific activity of 11C-Triflate-CH3I was more than 11CH3I. The quality control of [11C]-TKF was conformed to the "quality control of positron radiopharmaceuticals guiding principles" issued by SFDA. pH value of [11C]-TKF was neutral, sterile membrane permeability pressure was more than 0.4 MPa, the ethanol was less than 10%, bacteria and endotoxin were negative. The biodistribution of [11C]-TKF in normal mice revealed that [11C]-TKF was matabolized by gall bladder. What’s more, the brain uptake of [11C]-TKF was more excellent than [18F]-THK523.The acute toxicity of [11C]-TKF was negative. It is obvious that certain parameters can be determined solely by an in vivo administration. For example, of great importance with regard to visualization of NFTs in vivo is the extent of BBB penetration. Further research on [11C]-TKF are needed on the basis of preliminary studies. [11C]-TKF displayed excellent brain uptake and could be eluted quickly in normal mice. |
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