A probe for NIR-II imaging and multimodal analysis of early Alzheimer's disease by targeting CTGF.

To date, earlier diagnosis of Alzheimer's disease (AD) is still challenging. Recent studies revealed the elevated expression of connective tissue growth factor (CTGF) in AD brain is an upstream regulator of amyloid-beta (Aß) plaque, thus CTGF could be an earlier diagnostic biomarker of AD than Aß plaque. Herein, we develop a peptide-coated gold nanocluster that specifically targets CTGF with high affinity (KD ~ 21.9 nM). The probe can well penetrate the blood-brain-barrier (BBB) of APP/PS1 transgenic mice at early-stage (earlier than 3-month-old) in vivo, allowing non-invasive NIR-II imaging of CTGF when there is no appearance of Aß plaque deposition. Notably, this probe can also be applied to measuring CTGF on postmortem brain sections by multimodal analysis, including fluorescence imaging, peroxidase-like chromogenic imaging, and ICP-MS quantitation, which enables distinguishment between the brains of AD patients and healthy people. This probe possesses great potential for precise diagnosis of earlier AD before Aß plaque formation.

Loss of Bcl-3 regulates macrophage polarization by promoting macrophage glycolysis.

M1/M2 macrophage polarization plays an important role in regulating the balance of the microenvironment within tissues. Moreover, macrophage polarization involves the reprogramming of metabolism, such as glucose and lipid metabolism. Transcriptional coactivator B-cell lymphoma-3 (Bcl-3) is an atypical member of the IκB family that controls inflammatory factor levels in macrophages by regulating nuclear factor kappa B pathway activation. However, the relationship between Bcl-3 and macrophage polarization and metabolism remains unclear. In this study, we show that the knockdown of Bcl-3 in macrophages can regulate glycolysis-related gene expression by promoting the activation of the nuclear factor kappa B pathway. Furthermore, the loss of Bcl-3 was able to promote the interferon gamma/lipopolysaccharide-induced M1 macrophage polarization by accelerating glycolysis. Taken together, these results suggest that Bcl-3 may be a candidate gene for regulating M1 polarization in macrophages.

Detection of pTDP-43 via routine muscle biopsy: A promising diagnostic biomarker for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, pathologically characterized by TDP-43 aggregates. Recent evidence has been indicated that phosphorylated TDP-43 (pTDP-43) is present not only in motor neurons but also in muscle tissues. However, it is unclear whether testing pTDP-43 aggregation in muscle tissue would assist in the diagnosis of ALS. We propose three key questions: (i) Is aggregation of pTDP-43 detectable in routine biopsied muscles? (ii) Can detection of pTDP-43 aggregation discriminate between ALS and non-ALS patients? (iii) Can pTDP-43 aggregation be observed in the early stages of ALS? We conducted a diagnostic study comprising 2 groups: an ALS group in which 18 cases underwent muscle biopsy screened from a registered ALS cohort consisting of 802 patients and a non-ALS control group, in which we randomly selected 54 muscle samples from a biospecimen bank of 684 patients. Among the 18 ALS patients, 3 patients carried pathological GGGGCC repeats in the C9ORF72 gene, 2 patients carried SOD1 mutations, and 7 patients were at an early stage with only one body region clinically affected. The pTDP-43 accumulation could be detected in routine biopsied muscles, including biceps brachii, deltoid, tibialis anterior, and quadriceps. Abnormal aggregation of pTDP-43 was present in 94.4% of ALS patients (17/18) compared to 29.6% of non-ALS controls (16/54; p < 0.001). The pTDP-43 aggregates were mainly close to the sarcolemma. Using a semi-quantified pTDP-43 aggregates score, we applied a cut-off value of 3 as a diagnostic biomarker, resulting in a sensitivity of 94.4% and a specificity of 83.3%. Moreover, we observed that accumulation of pTDP-43 occurred in muscle tissues prior to clinical symptoms and electromyographic lesions. Our study provides proof-of-concept for the detection of pTDP-43 accumulation via routine muscle biopsy which may serve as a novel biomarker for diagnosis of ALS.

An NIR-II Probe with High PSMA Affinity Demonstrates an Unexpected Excellent Bone Imaging Ability.

(S)-3-(Carboxyformamido)-2-(3-(carboxymethyl)ureido)propanoic acid (EuK) is a known binder toward the prostate-specific membrane agent (PSMA) with strong affinity, making it a popular choice for prostate cancer medicine development. However, during the probe modification, a new EuK-based PSMA tetramer, Bone-1064, was discovered to have an unexpected and intense uptake in bone, which has not yet been reported in any previous studies yet. After administration, Bone-1064 allowed for high contrast visualization of the bone from surrounding tissues with a signal-to-background ratio of 10.22 at 24 h postinjection. In contrast, the tumor had a blurry contour, and the maximum tumor-to-normal-tissue ratio was only 2.22. Further imaging studies revealed that Bone-1064 binds specifically to hydroxyapatite in bone tissues, instead of PSMA. Overall, Bone-1064 is an excellent bone probe with a unique structure that can be used for NIR-II fluorescence imaging in animal models. Meanwhile, this modification study might also inspire further PSMA probe designations.

Structure-Activity Relationships of Cyano-substituted Indole Derivatives as Ligands for α-Synuclein Aggregates.

α-Synuclein (α-syn) is an essential biomarker for synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). The development of α-syn imaging probes is of great importance for understanding the pathogenesis mechanism and developing new therapies. In this study, we designed and synthesized a series of cyano-substituted indole derivatives and evaluated their potency to bind to α-syn fibrils by in vitro fibril binding assays. We carried out systematic structure-activity relationship (SAR) studies and obtained a promising candidate 51. The results showed that 51 bound to α-syn fibrils with the affinity of 17.4 ± 5.6 nM, and the biodistribution experiments in normal mice showed [125I]51 exhibited a moderate brain uptake of 3.57 ± 0.28% ID/g at 2 min after injection. In conclusion, the indole derivative [125I]51 showed initial potential as α-syn imaging probes, which needed further development.

Synthesis and Bioevaluation of 2-Styrylquinoxaline Derivatives as Tau-PET Tracers.

This study focused on designing and evaluating Tau-PET tracers for noninvasive positron emission computed tomography (PET) imaging of neurofibrillary tangles (NFTs), a hallmark pathology of Alzheimer's disease (AD). The tracers were synthesized with a 2-styrylquinoxaline scaffold and varying lengths of FPEG chains. The compound [18F]15, which had two ethoxy units, showed high affinity for recombinant K18-Tau aggregates (Ki = 41.48 nM) and the highest selectivity versus Aß1-42 aggregates (8.83-fold). In vitro autoradiography and fluorescent staining profiles further validated the binding of [18F]15 or 15 toward NFTs in brain sections from AD patients and Tau-transgenic mice. In normal ICR mice, [18F]15 exhibited an ideal initial brain uptake (11.21% ID/g at 2 min) and moderate washout ratio (2.29), and micro-PET studies in rats confirmed its ability to penetrate the blood-brain barrier with the peak SUV value of 1.94 in the cortex. These results suggest that [18F]15 has the potential to be developed into a useful Tau-PET tracer for early AD diagnosis and evaluation of anti-Tau therapeutics.

Synthesis, Preclinical Evaluation, and First-in-Human PET Study of [68Ga]-Labeled Biphenyl-Containing PSMA Tracers.

Radioisotope-labeled prostate-specific membrane antigen (PSMA) PET tracers have gained popularity in diagnosing prostate cancer (PCa). This study aimed to improve the affinity and tumor-targeting capabilities of new PSMA tracers by increasing the number of pharmacophores that specifically bind to PSMA. Using biphenyl as a core scaffold, we investigated the relationship among spacer segments, affinity, and pharmacokinetic properties. In preclinical PET studies on mice with 22Rv1 tumors, compared with [68Ga]Ga-PSMA-11 (SUVmax = 3.37), [68Ga]Ga-PSMA-D5 (Ki = 0.15) showed higher tumor uptake (SUVmax = 3.51) and lower renal uptake (T/K = 1.84). In the first-in-human study, [68Ga]Ga-PSMA-D5 effectively detected small PCa-associated lesions and distant metastases. The advantages of [68Ga]Ga-PSMA-D5 include high tumor uptake, straightforward synthesis, and labeling, making it a promising PSMA PET tracer. Furthermore, [68Ga]Ga-PSMA-D5 contains a DOTA chelator, allowing convenient labeling with therapeutic radionuclides such as 177Lu and 225Ac, providing the potential for targeted radioligand therapy in PCa.

Hydroxyethyl-Modified Cycloheptatriene-BODIPY Derivatives as Specific Tau Imaging Probes.

Near-infrared fluorescence (NIRF) imaging as an exquisite sensitive, high spatial-resolution, and real-time tool plays an important role in visualizing pathologies in the brain. In this study, we designed and synthesized a series of NIR probes of hydroxyethyl cycloheptatriene-BODIPY derivatives that have demonstrated strong binding specificity to native neurofibrillary tangles (NFTs) in Alzheimer's disease (AD) brain sections. The improved hydrophilicity of TNIR7-9 and TNIR7-11 resulted in faster clearance rates from healthy brains (4.2 and 10.9, respectively) compared to previously reported compounds. Furthermore, TNIR7-13, which features a fluorinated modification, exhibited a high binding affinity to Tau aggregates (Kd = 11.8 nM) and held promise for future PET studies.

Activated Microglia in the Early Stage of a Rat Model of Parkinson's Disease: Revealed by PET-MRI Imaging by [18F]DPA-714 Targeting TSPO.

In the past decades, translocator protein (TSPO) has been considered as an in vivo biomarker to measure the presence of neuroinflammatory reactions. In this study, expression of TSPO was quantified via [18F]DPA-714 positron emission tomography-magnetic resonance imaging (PET-MRI) to investigate the effects of microglial activation associated with motor behavioral impairments in the 6-hydroxydopamine (6-OHDA)-treated rodent model of Parkinson's disease (PD). [18F]FDG PET-MRI (for non-specific inflammation), [18F]D6-FP-(+)-DTBZ PET-MRI (for damaged dopaminergic (DA) neurons), post-PET immunofluorescence, and Pearson's correlation analyses were also performed. The time course of striatal [18F]DPA-714 binding ratio was elevated in 6-OHDA-treated rats during 1-3 weeks post-treatments, with peak TSPO binding in the 1st week. No difference between the bilateral striatum in [18F]FDG PET imaging were found. Moreover, an obvious correlation between [18F]DPA-714 SUVRR/L and rotation numbers was found (r = 0.434, *p = 0.049). No correlation between [18F]FDG SUVRR/L and rotation behavior was found. [18F]DPA-714 appeared to be a potential PET tracer for imaging the microglia-mediated neuroinflammation in the early stage of PD.

Synthesis and Preclinical Evaluation of 2-(4-[18F]Fluorophenyl)imidazo[1,2-h][1,7]naphthyridine ([18F]FPND-4): An Aza-Fused Tricyclic Derivative as Positron Emission Tomography Tracer for Neurofibrillary Tangle Imaging.

Tau accumulation is one of the predominant neuropathological biomarkers for in vivo diagnosis of Alzheimer's disease due to its high correlation with disease progression. In this study, we focused on the structure-activity relationship study of the substituent effect on the aza-fused tricyclic core imidazo[1,2-h][1,7]naphthyridine to screen 18F-labeled Tau tracers. Through a series of autoradiographic studies and biological evaluations, 4-[18F]fluorophenyl-substituted tracer [18F]13 ([18F]FPND-4) was identified as a promising candidate with high affinity to native Tau tangles (IC50 = 2.80 nM), few appreciable binding to Aß plaques and MAO-A/B. Validated by dynamic positron emission tomography (PET) imaging in rodents and rhesus monkey, [18F]13 displayed desirable brain uptake (SUV = 1.75 at 2 min), fast clearance (brain2min/60min = 5.9), minimal defluorination, and few off-target binding, which met the requirements of a Tau-specific PET radiotracer.

Design and Characterization of Squaramic Acid-Based Prostate-Specific Membrane Antigen Inhibitors for Prostate Cancer.

Prostate-specific membrane antigen (PSMA) overexpressed on prostate cancer (PCa) cells is a satisfactory theranostic target in PCa. To seek novel non-glutamate-urea-based PSMA inhibitors by the strategy of bioisosterism, 10 ligands were designed, synthesized, and characterized. Among them, ligands 17, 18, and 21-24 bearing the squaramic acid moiety proved to be potent PSMA inhibitors, with Ki values ranging from 0.40 to 2.49 nM, which are comparable or higher in inhibitory potency compared to previously reported glutamate-urea-based inhibitors. Docking studies of 15, 17, and 19 were carried out to explore their binding mode in the active site of PSMA. Two near-infrared (NIR) probes, 23 (λEM = 650 nm) and 24 (λEM = 1088 nm), displayed favorable in vivo NIR imaging and successful NIR-II image-guided tumor resection surgery in PSMA-positive tumor-bearing mice, which demonstrated the effectiveness of these new squaramic acid-based inhibitors.

Fluorine-18-Labeled Diaryl-azines as Improved ß-Amyloid Imaging Tracers: From Bench to First-in-Human Studies.

The deposition of ß-amyloid (Aß) in the brain is a pathologic hallmark of Alzheimer's disease (AD), appearing years before the onset of symptoms, and its detection is incorporated into clinical diagnosis. Here, we have discovered and developed a class of diaryl-azine derivatives for detecting Aß plaques in the AD brain using PET imaging. After a set of comprehensive preclinical assessments, we screened out a promising Aß-PET tracer, [18F]92, with a high binding affinity to the Aß aggregates, significant binding ability with the AD brain sections, and optimal brain pharmacokinetic properties in rodents and non-human primates. The first-in-human PET study declared that [18F]92 displayed low white matter uptake and could bind to Aß pathology for distinguishing AD from healthy control subjects. All these results support that [18F]92 might become a promising PET tracer for visualizing Aß pathology in AD patients.

Discovery and evaluation of aza-fused tricyclic derivatives for detection of Tau pathology in Alzheimer's disease.

For various neurodegenerative diseases, including Alzheimer's disease (AD), the abnormal aggregation of Tau is not only the predominant contributing factor but also a major biomarker for disease diagnosis. In this study, a series of aza-fused tricyclic derivatives were designed and synthesized. By changing the position and number of nitrogen atoms on the fused tricyclic core, the imidazonaphthyridine scaffold was screened and reported for the first time which could potentially detect Tau aggregates. Through a series of in vitro and in vivo biological evaluations, probe [125I]5 possessed exceptional binding affinity (IC50 = 1.63 nM) to neurofibrillary tangles in the AD brain, high selectivity over Aß plaques (23.4-fold), clean off-target profile to monoamine oxidase A/B (MAO-A/B), and suitable pharmacokinetics (initial brain uptake = 3.22% ID/g).

D-π-A-Based Trisubstituted Alkenes as Environmentally Sensitive Fluorescent Probes to Detect Lewy Pathologies.

Lewy pathologies, which mainly consist of insoluble α-synuclein (α-syn) aggregates, are the hallmarks of Parkinson's disease and many other neurodegenerative diseases termed "synucleinopathies". Detection of Lewy pathologies with optical methods is of interest for preclinical studies, while the α-syn fluorescent probe is still in great demand. By rational design, we obtained a series of D-π-A-based trisubstituted alkenes with acceptable optical properties and high binding affinities to α-syn fibrils. Among these probes, FPQXN and TQXN-2 exhibited high binding affinities (6 and 8 nM, respectively), significant fluorescence enhancements (17.2- and 26.6-fold, respectively), and satisfying quantum yields (36.5% and 10.4%, respectively), which met the need for the in vitro neuropathological staining of Lewy pathologies in the PD brain sections. In addition, TQXN-2 showed great potential in fluorescent discrimination of Lewy pathologies and Aß plaques. Our research provides flexible tools for in vitro detection of α-syn aggregates and offers new structural frameworks for the further development of α-syn fluorescent probes.

Fused Cycloheptatriene-BODIPY Is a High-Performance Near-Infrared Probe to Image Tau Tangles.

Neurofibrillary tangles (NFTs), which are composed of abnormally hyperphosphorylated Tau, are one of the main pathologic hallmarks of Alzheimer's disease and other tauopathies. The fluorescent imaging probes currently used to target NFTs cannot distinguish them well from ß-amyloid plaques, thus limiting their utility to diagnose diseases. Here, we developed a fused cycloheptatriene-BODIPY derivative (TNIR7-1A) that displays properties favorable for near-infrared (NIR) imaging with high affinity and specificity to NFTs in vitro. In addition, TNIR7-1A effectively penetrated the blood-brain barrier and clearly distinguished tauopathy in transgenic mice (rTg4510) from control mice using NIR fluorescence imaging in vivo. The sensitivity and specificity of TNIR7-1A for NFTs were confirmed ex vivo by fluorescence staining of the tauopathy mouse model, while molecular docking studies indicated that TNIR7-1A bound to NFTs through hydrophobic interactions. These results suggest that TNIR7-1A can act as a high-performance probe to detect NFTs in vitro and in vivo selectively.

A Preclinical Study of an 125I-Labeled PSMA Ligand for Prostate-Cancer Puncture.

PURPOSE: Prostate cancer (PCa) is characterized by high expression of prostate-specific 1membrane antigen (PSMA), a type II transmembrane protein. Prostate-specific membrane antigen positron emission tomography (PSMA PET) has high sensitivity and specificity and can therefore be potentially used to detect PCa. Exploiting the advantages of PSMA PET imaging, in this study, we aim to develop a novel radiopharmaceutical to facilitate biopsy punching of PCa. METHODS: We synthesized a high-affinity radiopharmaceutical of PSMA (125I-PSMA-7). We evaluated the properties of 125I-PSMA-7, including the purity, stability, affinity, partition coefficient, and toxicity. (PSMA+) 22Rv1 and (PSMA-) PC3 cell lines were used to evaluate 125I-PSMA-7 in vitro. BALB/c nude mice bearing 22Rv1 and PC3 xenografts were used for biodistribution and imaging. The uptake of the main organs was evaluated in vivo using single photon emission computed tomography (SPECT). RESULTS: 125I-PSMA-7 had a purity of 99.6% and remained stable for seven days and was therefore always safe to use. 125I-PSMA-7 had a Ki of 4.037 × 10-11 and a partition coefficient of -1.80. The results of in vitro cellular experiments showed a high uptake by 22Rv1 cells (ranging from 2.88 ± 0.14 IA%/106 at 5 min to 61.98 ± 3.43 IA%/106 at 24 h, where the internalization was 46.1% at 1 h and 88.06% at 24 h). However, the uptake of PC3 cells was very low (ranging from 0.34 ± 0.08 IA%/106 at 5 min to 1.60 ± 0.15 IA%/106 at 24 h). The tumors' uptake of 125I-PSMA-7 ranged from 9.02 ± 0.30 ID%/g at 1 h to 4.11 ± 1.04 ID%/g at 7 d and the tumor/muscle ratios and tumor/blood ratios increased over time. In addition, we used γ-counter to measure cpm per milligram of tumor and muscle on days 4 and 7. The background on day 4 is 42 cpm and the tumor is 1739 cpm/mg and the muscle is 45 cpm/mg, and the background on day 7 is 74 cpm and the tumor is 1404cpm/mg and the muscle is 32 cpm/mg. At 1 h post-injection, the high uptake of 125I-PSMA-7 resulted in clear delineation of 22Rv1-derived tumors upon imaging. By comparison, 22Rv1-blocking mice took up less 125I-PSMA-7. CONCLUSIONS: These results show that 125I-PSMA-7 is a promising radiotracer that could be used to puncture the prostate. 125I-PSMA-7 could be applied to targeted biopsy, reducing the need for saturated biopsy.

Rational design of molecular rotor-based fluorescent probes with bi-aromatic rings for efficient in vivo detection of amyloid-ß plaques in Alzheimer's disease.

The presence of Aß plaques in the brain is a hallmark of Alzheimer's disease. Here, we designed and synthesized a series of molecular rotors with various bi-aromatic rings and investigated their applications as near-infrared (NIR) probes for Aß plaques. We found that the interaction with Aß aggregates hindered the rotational freedom of the molecular rotors, which brought about a noticeable enhancement in fluorescence intensity. Among them, probe 4b (Kd = 8.5 nM) with a phenyl-pyridine ring showed a 98-fold increase in fluorescence intensity upon binding with Aß aggregates. In addition, 4b could identify Aß plaques in brain sections of both a transgenic (Tg) mouse and AD patients. Furthermore, 4b could readily penetrate the mouse blood-brain barrier (brain2min = 10.11% ID/g) and washed out rapidly. Finally, the NIR imaging with Tg mice confirmed the practical application of 4b in detecting Aß plaques in vivo. Altogether, our work widens the landscape of Aß NIR probes and offers a new tool for Aß detection.

In vivo imaging of tau deposition in Alzheimer's disease using both [18F]-THK5317 and [18F]-S16: A pilot human study.

Objective: To evaluate the effectiveness of a new tracer (S)-1-(4-(6-(dimethylamino)quinoxalin-2-yl)phenoxy)-3-fluoropropan-2-ol ([18F]-S16), in distinguishing patients with AD from HCs. Methods: Paired [18F]-S16 and [18F]-THK5317 scans were acquired in five patients with AD, six HCs, one subject with a semantic variant of primary progressive aphasia (sv-PPA) and one subject with probable progressive supranuclear palsy (PSP). Dynamic PET scanning was performed over 90 min after injection of the tracers. Standardized uptake values (SUV) and cortical-to-cerebellum standardized uptake value ratios (SUVRs) were used for tau deposition semi-quantization. A voxel-based analysis was employed to assess the uptake difference between populations. Results: [18F]-S16 exhibited excellent blood-brain-barrier penetration. AD patients showed increased cortical [18F]-THK5317 and [18F]-S16 binding. Compared to HCs, AD patients showed significantly increased cortical [18F]-S16 uptake in the bilateral occipital cortex, posterior cingulated cortex/precuneus, and lateral frontal cortex. Notable [18F]-S16 uptake was observed in the basal ganglia and brainstem compared to the neocortex. A substantial [18F]-S16 signal was detected in the basal ganglia and midbrain in a patient with probable PSP and in the bilateral anterior temporal cortex in a sv-PPA patient. Conclusion: [18F]-S16 might be of help to detect tau protein in vivo.

Discovery and development of brain-penetrant 18F-labeled radioligands for neuroimaging of the sigma-2 receptors.

We have discovered and synthesized a series of indole-based derivatives as novel sigma-2 (σ 2) receptor ligands. Two ligands with high σ 2 receptor affinity and subtype selectivity were then radiolabeled with F-18 in good radiochemical yields and purities, and evaluated in rodents. In biodistribution studies in male ICR mice, radioligand [18F]9, or 1-(4-(5,6-dimethoxyisoindolin-2-yl)butyl)-4-(2-[18F]fluoroethoxy)-1H-indole, was found to display high brain uptake and high brain-to-blood ratio. Pretreatment of animals with the selective σ 2 receptor ligand CM398 led to significant reductions in both brain uptake (29%-54%) and brain-to-blood ratio (60%-88%) of the radioligand in a dose-dependent manner, indicating high and saturable specific binding of [18F]9 to σ 2 receptors in the brain. Further, ex vivo autoradiography in male ICR mice demonstrated regionally heterogeneous specific binding of [18F]9 in the brain that is consistent with the distribution pattern of σ 2 receptors. Dynamic positron emission tomography imaging confirmed regionally distinct distribution and high levels of specific binding for [18F]9 in the rat brain, along with appropriate tissue kinetics. Taken together, results from our current study indicated the novel radioligand [18F]9 as the first highly specific and promising imaging agent for σ 2 receptors in the brain.