Trait-anxiety and glial-related neuroinflammation of the amygdala and its associated regions in Alzheimer's disease: A significant correlation.

Background: Positron emission tomography, which assesses the binding of translocator protein radiotracers, 11C-DPA-713, may be a sensitive method for determining glial-mediated neuroinflammation levels. This study investigated the relationship between regional 11C-DPA713 binding potential (BPND) and anxiety in patients with Alzheimer's disease (AD) continuum. Methods: Nineteen patients with AD continuum determined to be amyloid-/p-tau 181-positive via cerebrospinal fluid analysis were included in this cross-sectional study (mild cognitive impairment [MCI, n = 5] and AD [n = 14]). Anxiety was evaluated using the State-Trait Anxiety Inventory (STAI). A whole-brain voxel-based analysis was performed to examine the relationship between 11C-DPA-713-BPND values at each voxel and the STAI score. Stepwise multiple regression analysis was performed to determine the predictors of STAI scores using independent variables, including 11C-DPA-713-BPND values within significant clusters. 11C-DPA-713-BPND values were compared between patients with AD continuum with low-to-moderate and high STAI scores. Results: Voxel-based analysis revealed a positive correlation between trait anxiety severity and 11C-DPA713-BPND values in the centromedial amygdala and the left inferior occipital area [P < 0.001 (uncorrected) at the voxel-level]. 11C-DPA713-BPND values in these regions were a strong predictor of the STAI trait anxiety score. Specifically, patients with AD continuum and high trait anxiety had increased 11C-DPA713-BPND values in these regions. Conclusions: The amygdala-occipital lobe circuit influences the control of emotional generation, and disruption of this network by AD pathology-induced inflammation may contribute to the expression of anxiety. Our findings suggest that suppression of inflammation can help effectively treat anxiety by attenuating damage to the amygdala and its associated areas.

A novel PET probe to selectively image heat shock protein 90α/ß isoforms in the brain.

BACKGROUND: Heat shock proteins (HSPs) are present throughout the brain. They function as molecular chaperones, meaning they help with the folding and unfolding of large protein complexes. These chaperones are vital in the development of neuropathological conditions such as Alzheimer's disease and Lewy body disease, with HSP90, a specific subtype of HSP, playing a key role. Many studies have shown that drugs that inhibit HSP90 activity have beneficial effects in the neurodegenerative diseases. Therefore, HSP90 PET imaging ligand can be used effectively to study HSP90 in neurodegenerative diseases. Among four HSP90 isoforms, two cytosolic isoforms (HSP90α and HSP90ß) thought to be involved in the structural homeostasis of the proteins related to the neurodegenerative diseases. Currently, no useful PET imaging ligands selectively targeting the two cytosolic isoforms of HSP90 have been available yet. RESULTS: In this study, we developed a novel positron emission tomography (PET) imaging ligand, [11C]BIIB021, by 11C-radiolabeling (a positron emitter with a half-life of 20.4 min) 6-Chloro-9-[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]-9H-purin-2-amine (BIIB021), an inhibitor with a high affinity for and selectivity to HSP90α and HSP90ß. [11C]BIIB021 was synthesized with a high yield, molar activity and radiochemical purity. [11C]BIIB021 showed a high binding affinity for rat brain homogenate as well as human recombinant HSP90α and HSP90ß proteins. Radioactivity was well detected in the rat brain (SUV 1.4). It showed clear specific binding in PET imaging of healthy rats and autoradiography of healthy rat and human brain sections. Radiometabolite was detected in the brain, however, total distribution volume was well quantified using dual-input graphical model. Inhibition of p-glycoprotein increased brain radioactivity concentrations. However, total distribution volume values with and without p-glycoprotein inhibition were nearly the same. CONCLUSIONS: We have developed a new PET imaging agent, [11C]BIIB021, specifically targeting HSP90α/ß. We have been successful in synthesizing [11C]BIIB021 and in vitro and in vivo imaging HSP90α/ß. However, the quantification of HSP90α/ß is complicated by the presence of radiometabolites in the brain and the potential to be a substrate for p-glycoprotein. Further efforts are needed to develop radioligand suitable for imaging of HSP90α/ß.

Synthesis and evaluation of a novel PET ligand, a GSK'963 analog, aiming at autoradiography and imaging of the receptor interacting protein kinase 1 in the brain.

BACKGROUND: Receptor interacting protein kinase 1 (RIPK1) is a serine/threonine kinase, which regulates programmed cell death and inflammation. Recently, the involvement of RIPK1 in the pathophysiology of Alzheimer's disease (AD) has been reported; RIPK1 is involved in microglia's phenotypic transition to their dysfunctional states, and it is highly expressed in the neurons and microglia in the postmortem brains in AD patients. They prompt neurodegeneration leading to accumulations of pathological proteins in AD. Therefore, regulation of RIPK1 could be a potential therapeutic target for the treatment of AD, and in vivo imaging of RIPK1 may become a useful modality in studies of drug discovery and pathophysiology of AD. The purpose of this study was to develop a suitable radioligand for positron emission tomography (PET) imaging of RIPK1. RESULTS: (S)-2,2-dimethyl-1-(5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)propan-1-one (GSK'963) has a high affinity, selectivity for RIPK1, and favorable physiochemical properties based on its chemical structure. In this study, since 11C-labeling (half-life: 20.4 min) GSK'963 retaining its structure requiring the Grignard reaction of tert-butylmagnesium halides and [11C]carbon dioxide was anticipated to give a low yield, we decided instead to 11C-label a GSK'963 analog ((S)-2,2-dimethyl-1-(5-(m-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)propan-1-one, GG502), which has a high RIPK1 inhibitory activity equivalent to that of the original compound GSK'963. Thus, we successfully 11C-labeled GG502 using a Pd-mediated cross-coupling reaction in favorable yields (3.6 ± 1.9%) and radiochemical purities (> 96%), and molar activity (47-115 GBq/µmol). On autoradiography, radioactivity accumulation was observed for [11C]GG502 and decreased by non-radioactive GG502 in the mouse spleen and human brain, indicating the possibility of specific binding of this ligand to RIPK1. On brain PET imaging in a rhesus monkey, [11C]GG502 showed a good brain permeability (peak standardized uptake value (SUV) ~3.0), although there was no clear evidence of specific binding of [11C]GG502. On brain PET imaging in acute inflammation model rats, [11C]GG502 also showed a good brain permeability, and no significant increased uptake was observed in the lipopolysaccharide-treated side of striatum. On metabolite analysis in rats at 30 min after administration of [11C]GG502, ~55% and ~10% of radioactivity was from unmetabolized [11C]GG502 in the brain and the plasma, respectively. CONCLUSIONS: We synthesized and evaluated a 11C-labeled PET ligand based on the methylated analog of GSK'963 for imaging of RIPK1 in the brain. Although in autoradiography of the resulting [11C]GG502 indicated the possibility of specific binding, the actual PET imaging failed to detect any evidence of specific binding to RIPK1 despite its good brain permeability. Further development of radioligands with a higher binding affinity for RIPK1 in vivo and more stable metabolite profiles compared with the current compound may be required.

Noradrenaline transporter PET reflects neurotoxin-induced noradrenaline level decrease in the rat hippocampus.

BACKGROUND: The neuropathological changes of early Alzheimer's disease (AD) include neurodegenerative loss of noradrenaline neurons in the locus coeruleus with decreasing noradrenaline availability in their projection areas such as the hippocampus. This diminishing noradrenaline availability is thought to play an important role pathophysiologically in the development of cognitive impairment in AD, because noradrenaline is not only essential for maintaining cognitive functions such as memory, learning and attention, but also its anti-inflammatory action, where its lack is known to accelerate the progression of AD in the mouse model. Therefore, the availability of in vivo biomarkers of the integrity of noradrenaline neurons may be beneficial for furthering our understanding of the role played by the noradrenaline system in the progressive cognitive dysfunction seen in AD patients. In this study, we investigated if PET imaging of noradrenaline transporters can predict the level of noradrenaline in the brain. Our hypothesis was PET measured noradrenaline transporter densities could predict the level of noradrenaline concentrations in the rat hippocampus after lesioning of noradrenaline neurons in this region. RESULTS: We chemically lesioned the hippocampus of rats (n = 15) by administering a neurotoxin, DSP-4, in order to selectively damage axonal terminals of noradrenergic neurons. These rats then underwent PET imaging of noradrenaline transporters using [11C]MRB ((S,S)-[11C]Methylreboxetine). To validate our hypothesis, postmortem studies of brain homogenates of these rats were performed to measure both noradrenaline transporter and noradrenaline concentrations. [11C]MRB PET showed decreased noradrenaline transporter densities in a DSP-4 dose-dependent manner in the hippocampus of these rats. In turn, these PET measured noradrenaline transporter densities correlated very well with in vitro measured noradrenaline concentrations as well as in vitro transporter densities. CONCLUSIONS: [11C]MRB PET may be used as an in vivo biomarker of noradrenaline concentrations in the hippocampus of the neurodegenerating brain. Further studies appear warranted to extend its applicability to AD studies.

Neuroimaging biomarkers of glial activation for predicting the annual cognitive function decline in patients with Alzheimer's disease.

BACKGROUND: Glial activation is central to the pathogenesis of Alzheimer's disease (AD). However, researchers have not demonstrated its relationship to longitudinal cognitive deterioration. We aimed to compare the prognostic effects of baseline positron emission tomography (PET) imaging of glial activation and amyloid/tau pathology on the successive annual cognitive decline in patients with AD. METHODS: We selected 17 patients diagnosed with mild cognitive impairment or AD. We assessed the annual changes in global cognition and memory. Furthermore, we assessed the predictive effects of baseline amyloid and tau pathology indicated by cerebrospinal fluid (CSF) concentrations and PET imaging of glial activation (11C-DPA-713-binding potential in the area of Braak 1-3 [11C-DPA-713-BPND]) on global cognition and memory using a stepwise regression analysis. RESULTS: The final multiple regression model of annual changes in global cognition and memory scores included 11C-DPA-713-BPND as the predictor. The CSF Aß42/40 ratios and p-tau concentrations were removed from the final model. In stepwise Bayesian regression analysis, the Bayes factor-based model comparison suggested that the best model included 11C-DPA-713-BPND as the predictor of decline in global cognition and memory. CONCLUSIONS: Translocator protein-PET imaging of glial activation is a stronger predictor of AD clinical progression than the amount of amyloid/tau pathology measured using CSF concentrations. Glial activation is the primary cause of tau-induced neuronal toxicity and cognitive deterioration, thereby highlighting the potential of blocking maladaptive microglial responses as a therapeutic strategy for AD treatment.

Development of a novel PET ligand, [11C]GO289 targeting CK2 expressed in the brain.

Positron emission tomography (PET) is a powerful imaging tool that enables early in vivo detection of Alzheimer's disease (AD). For this purpose, various PET ligands have been developed to image ß-amyloid and tau protein aggregates characteristically found in the brain of AD patients. In this study, we initiated to develop another type of PET ligand that targets protein kinase CK2 (formerly termed as casein kinase II), because its expression level is known to be altered in postmortem AD brains. CK2 is a serine/threonine protein kinase, an important component of cellular signaling pathways that control cellular degeneration. In AD, the CK2 level in the brain is thought to be elevated by its involvement in both phosphorylation of proteins such as tau and neuroinflammation. Decreased CK2 activity and expression levels lead to ß-amyloid accumulation. In addition, since CK2 also contributes to the phosphorylation of tau protein, the expression level and activity of CK2 is expected to undergo significant changes during the progression of AD pathology. Furthermore, CK2 could act as a potential target for modulating the inflammatory response in AD. Therefore, PET imaging targeting CK2 expressed in the brain could be a useful another imaging biomarker for AD. We synthesized and radiolabeled a CK2 inhibitor, [11C]GO289, in high yields from its precursor and [11C]methyl iodide under basic conditions. On autoradiography, [11C]GO289 specifically bound to CK2 in both rat and human brain sections. On baseline PET imaging, this ligand entered and rapidly washed out of the rat brain with its peak activity rather being small (SUV < 1.0). However, on blocking, there was no detectable CK2 specific binding signal. Thus, [11C]GO289 may be useful in vitro but not so in vivo in its current formulation. The lack of detectable specific binding signal in the latter may be due to a relatively high component of nonspecific binding signal in the overall rather weak PET signal, or it may also be related to the known fact that ATP can competitively binds to subunits of CK2, reducing its availability for this ligand. In the future, it will be necessary for PET imaging of CK2 to try out different non-ATP competitive formulations of CK2 inhibitor that can also provide significantly higher in vivo brain penetration.

Sasa veitchii extracts protect phenytoin-induced cell proliferation inhibition in human lip mesenchymal cells through modulation of miR-27b-5p.

A cleft lip, with or without a cleft palate, is a common birth defect caused by environmental factors or genetic mutations. Environmental factors, such as pharmaceutical exposure in pregnant women, are known to induce cleft lip, with or without cleft palate in the child. This study aimed to investigate the protective effect of Sasa veitchii extract (SE) on phenytoin-induced inhibition of cell proliferation in human lip mesenchymal cells (KD cells) and human embryonic palatal mesenchymal cells (HEPM cells). We demonstrated that cell proliferation was inhibited by phenytoin in a dose-dependent manner in both KD and HEPM cells. Co-treatment with SE restored phenytoin-induced toxicity in KD cells but did not protect HEPM cells against phenytoin-induced toxicity. Several microRNAs (miR-27b, miR-133b, miR-205, miR-497-5p, and miR-655-3p) is reported to associate with cell proliferation in KD cells. We measured the seven kinds of microRNAs (miR27b-3p, miR-27b-5p, miR-133b, miR-205-3p, miR-205-5p, miR-497-5p, and miR-655-3p) and found that SE suppressed miR-27b-5p induced by phenytoin in KD cells. Furthermore, co-treatment with SE enhanced the expression of miR-27b-5p downstream genes (PAX9, RARA, and SUMO1). These results suggest that SE protects phenytoin-induced cell proliferation inhibition by modulating miR-27b-5p.

11C-Labeling of acyclic retinoid peretinoin by rapid C-[11C]methylation to disclose novel brain permeability and central nervous system activities hidden in antitumor agent.

Recently, retinoid actions on the central nervous system (CNS) have attracted considerable attention from the perspectives of brain disease diagnosis and drug development. Firstly, we successfully synthesized [11C]peretinoin esters (methyl, ethyl, and benzyl) using a Pd(0)-mediated rapid C-[11C]methylation of the corresponding stannyl precursors without geometrical isomerization in 82%, 66%, and 57% radiochemical yields (RCYs). Subsequent hydrolysis of the 11C-labeled ester produced [11C]peretinoin in 13 ± 8% RCY (n = 3). After pharmaceutical formulation, the resulting [11C]benzyl ester and [11C]peretinoin had high radiochemical purity (>99% each) and molar activities of 144 and 118 ± 49 GBq µmol-1 at total synthesis times of 31 min and 40 ± 3 min, respectively. Rat brain PET imaging for the [11C]ester revealed a unique time-radioactivity curve, suggesting the participation of the acid [11C]peretinoin for the brain permeability. However, the curve of the [11C]peretinoin rose steadily after a shorter time lag to reach 1.4 standardized uptake value (SUV) at 60 min. These various phenomena between the ester and acid became more pronounced in the monkey brain (SUV of > 3.0 at 90 min). With the opportunity to identify high brain uptake of [11C]peretinoin, we discovered CNS activities of a drug candidate called peretinoin, such as the induction of a stem-cell to neuronal cell differentiation and the suppression of neuronal damages.

Involvement of inflammation in the medial temporal region in the development of agitation in Alzheimer's disease: an in vivo positron emission tomography study.

BACKGROUND: The evaluation of 11 C-DPA-713 binding using positron emission tomography for quantifying the translocator protein can be a sensitive approach in determining the level of glial activation induced by neuroinflammation. Herein, we aimed to investigate the relationship between regional 11 C-DPA713-binding potential (BPND ) and neuropsychiatric symptoms (NPS) in amyloid-positive Alzheimer's disease (AD) patients. METHODS: Fifteen AD patients were enrolled in this study. Correlations were evaluated between the 11 C-DPA713-BPND and Neuropsychiatric Inventory Questionnaire (NPI-Q) scores, including scores in its four domains: agitation, psychosis, affective, and apathy. 11 C-DPA713-BPND values were compared between groups with and without the neuropsychiatric symptoms for which a relationship was observed in the abovementioned correlation analysis. RESULTS: A positive correlation was found between the severity of agitation and 11 C-DPA713-BPND in the Braak 1-3 area, including the amygdala, hippocampal and parahippocampal regions, and lingual and fusiform areas. An increase in the 11 C-DPA713-BPND was observed in AD patients with agitation. We did not find any significant effects of possible confounding factors, such as age, duration of illness, education, gender, Mini-Mental State Examination score, cerebrospinal fluid amyloid ß 42/40 ratio, and apolipoprotein E4 positivity, on either the 11 C-DPA713-BPND or agitation score. CONCLUSIONS: Neuroinflammation in the medial temporal region and its neighbouring area was shown to be associated with the development of agitation symptoms in AD patients. Our findings extend those of previous studies showing an association between some NPS and inflammation, suggesting that immunologically based interventions for agitation can serve as an alternative treatment for dementia.

Estimation of blood-based biomarkers of glial activation related to neuroinflammation.

Background: Neuroinflammation is a well-known feature of Alzheimer's disease (AD), and a blood-based test for estimating the levels of neuroinflammation would be expected. In this study, we examined and validated a model using blood-based biomarkers to predict the level of glial activation due to neuroinflammation, as estimated by 11C-DPA-713 positron emission tomography (PET) imaging. Methods: We included 15 patients with AD and 10 cognitively normal (CN) subjects. Stepwise backward deletion multiple regression analysis was used to determine the predictors of the TSPO-binding potential (BPND) estimated by PET imaging. The independent variables were age, sex, diagnosis, apolipoprotein E4 positivity, body mass index and the serum concentration of blood-based biomarkers, including monocyte chemotactic protein 1 (MCP-1), fractalkine, chitinase 3-like protein-1 (CHI3L1), soluble triggering receptor expressed on myeloid cells 2 (sTREM2), and clusterin. Results: Sex, diagnosis, and serum concentrations of MCP1 and sTREM2 were determined as predictors of TSPO-BPND in the Braak1-3 area. The serum concentrations of MCP1 and sTREM2 correlated positively with TSPO-BPND. In a leave one out (LOO) cross-validation (CV) analysis, the model gave a LOO CV R2 of 0.424, which indicated that this model can account for approximately 42.4% of the variance of brain TSPO-BPND. Conclusions: We found that the model including serum MCP-1 and sTREM2 concentration and covariates of sex and diagnosis was the best for predicting brain TSPO-BPND. The detection of neuroinflammation in AD patients by blood-based biomarkers should be a sensitive and useful tool for making an early diagnosis and monitoring disease progression and treatment effectiveness.

[11C]NCGG401, a novel PET ligand for imaging of colony stimulating factor 1 receptors.

Colony-stimulating factor 1 receptors (CSF1R) are expressed exclusively on microglia in the central nervous system. The receptors regulate immune responses by controlling the survival and activity of microglia and are intricately involved in the pathophysiology of Alzheimer's disease. In this study, we developed [11C]NCGG401, a positron emission tomography (PET) ligand, targeting for CSF1R as an imaging biomarker for microglial pathophysiology in Alzheimer's disease. NCGG401 showed a high potency to inhibit human CSF1R kinase activity and a high binding affinity to human CSF1R. PET imaging with [11C]NCGG401 in healthy rats showed a good brain permeability. Furthermore, the specific binding component was determined by postmortem autoradiography in rat brain and human hippocampal sections. The knowledge of the characteristics of [11C]NCCC401, our initial CSF1R compound, we have obtained may be useful for further development and optimization of CSF1R radioligands for PET imaging of microglia.

Kinetic modeling and non-invasive approach for translocator protein quantification with 11C-DPA-713.

INTRODUCTION: 11C-DPA-713 is a positron emission tomography (PET) radiotracer developed for imaging the expression of the translocator protein (TSPO) in glial cells, which is considered to be a marker of the neuroinflammatory burden. This study investigated the pharmacokinetic profile of 11C-DPA-713 and evaluated kinetic modeling and non-invasive TSPO quantification using dynamic PET imaging data in the Alzheimer's disease (AD) and cognitive normal (CN) participants. METHODS: Eleven patients with AD and 6 CN participants were examined using dynamic 11C-DPA-713 PET imaging for 60 min with arterial blood sampling. Time-activity curves were calculated from the cerebellum and three composite regions of interest (ROIs), according to the anatomical definitions of Braak's stages 1 to 3, stage 4, stage 5, and stage 6 that correspond to the pathological stages of tangle deposition. The total distribution volume (VT) was evaluated using compartmental modeling and graphical analysis. Reference region-based methods were implemented using an optimal area that was assumed to be void of the radiotracer target as reference tissue. RESULTS: The concentration of radioactivity in plasma demonstrated rapid clearance. 11C-DPA-713 peaked rapidly in the gray matter. Compartmental modeling resulted in a good fit, and the one-tissue model with estimated blood volume correction (1Tv) showed the best performance. The estimated VT obtained from the graphical plasma methods was highly correlated with that obtained from 1Tv. Reference region-based analysis was conducted using the Braak 6 area as the reference region, and the estimated non-displaceable binding potential was highly correlated with that obtained from 1Tv. CONCLUSION: 11C-DPA-713 possesses properties suitable for TSPO quantification with PET imaging. The Braak 6 area was shown to be a useful reference region in the patients with AD and the CN participants, and non-invasive reference tissue models using the Braak 6 area as a reference region can be employed for TSPO quantification with 11C-DPA-713-PET imaging as an alternative to the invasive compartmental model.

Biocontrol Activity of Nonpathogenic Strains of Fusarium oxysporum: Colonization on the Root Surface to Overcome Nutritional Competition.

Fusarium oxysporum is a soil-borne fungal pathogen that causes vascular wilts in a wide variety of crops. Certain nonpathogenic strains of F. oxysporum are known to protect crops against F. oxysporum pathogens. We assessed the biocontrol activities of nonpathogenic mutants of F. oxysporum ff. spp. melonis and lycopersici generated by disruption of the FOW2 gene, which encodes a Zn(II)2Cys6-type transcriptional regulator essential for their pathogenicity. Pre-inoculation of melon or tomato roots with strain ΔFOW2 conidia markedly reduced disease incidence caused by the parental wild-type strain in a concentration-dependent manner of conidial suspensions of ΔFOW2 strains. The biocontrol effect caused by the ΔFOW2 pre-inoculation lasted for at least 7 days. Pre-inoculation of melon roots with the wild-type or ΔFOW2 strain of F. oxysporum f. sp. lycopersici and nonpathogenic F. oxysporum strain also led to biocontrol activity against F. oxysporum f. sp. melonis, indicating that the biocontrol activity of ΔFOW2 strains is due to its nonpathogenic nature, not to the FOW2 disfunction. Conidial germination and hyphal elongation of only the wild-type strain were inhibited on melon root surface pre-inoculated with conidia of strains nonpathogenic to melon plants. Expression of defense-related genes was not significantly induced in roots and aboveground parts of melon seedlings preinoculated with ΔFOW2 conidia. Carbon source competition assay showed that nonpathogenic strains competed with the wild-type strain for a carbon source in soil. Strain ΔFOW2 also competed with the oomycete pathogen Pythium aphanidermatum for carbon source and protected melon plants from P. aphanidermatum. Our results suggest that the biocontrol activity of the nonpathogenic F. oxysporum strains used in this study mainly depends on their extensive colonization of the root surface and outcompeting pathogens for nutrients.

Brain pharmacokinetics and biodistribution of 11C-labeled isoproterenol in rodents.

INTRODUCTION: Isoproterenol is a non-selective ß receptor agonist, which is a drug approved for bradycardia and bronchial asthma in many countries. Recently, isoproterenol has been reported to have the potential as a drug for the treatment of Alzheimer's disease by inhibiting the aggregation of tau protein. Isoproterenol is a highly potent drug causing increases in heart rates even when its plasma concentration is very low. Thus, it is critical to know if potentially effective therapeutic levels of isoproterenol can be achieved, maintaining safe plasma levels without any untoward pharmacological effects. The purpose of the study is to investigate the brain pharmacokinetics and biodistribution of 11C-labeled isoproterenol in rodents. METHODS: We performed positron emission tomography (PET) brain imaging and biodistribution studies of [11C]isoproterenol. 120-min scans with arterial blood sampling were performed in rats. Additionally, plasma and brain homogenates were analyzed with radio-HPLC to characterize its metabolite profiles. As a measure of [11C]isoproterenol brain uptake, total distribution volumes were determined by a pharmacokinetic compartment model. Biodistribution of [11C]isoproterenol was investigated in mice at six-time points from 1-min to 90-min after injection. RESULTS: We found a modest brain uptake of [11C]isoproterenol. Its brain pharmacokinetics showed that the concentration of isoproterenol in the brain at equilibrium was about two-fold higher than in the plasma (total distribution volumes 2.0 ± 0.2 cm3/mL). Only unmetabolized isoproterenol was detected in the brain at 30 min after injection, although isoproterenol was rapidly metabolized in plasma. The biodistribution study showed that isoproterenol and its metabolite are excreted mainly via the urinary system. CONCLUSIONS, ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: In this study, we have shown that rat brain concentrations of isoproterenol are only two-fold of that in plasma at equilibrium. If the brain pharmacokinetics are similar in the human brain, it may be difficult to achieve potentially therapeutic levels of this drug safely in humans. Further studies appear warranted to investigate the brain pharmacokinetics in humans with PET using [11C]isoproterenol.

Synthesis of (R,S)-isoproterenol, an inhibitor of tau aggregation, as an 11C-labeled PET tracer via reductive alkylation of (R,S)-norepinephrine with [2-11C]acetone.

(R,S)-Isoproterenol inhibits the formation of toxic granular tau oligomers associated with neuronal loss and development of cognitive disorders, and is an attractive drug candidate for Alzheimer's disease. To elucidate its behavior in the brain by positron emission tomography, we synthesize (R,S)-[11C]isoproterenol by reductive alkylation of (R,S)-norepinephrine with [2-11C]acetone, which was in turn synthesized in situ under improved conditions afforded a decay-corrected radiochemical yield of 54%. The reductive alkylation using NaBH(OAc)3 as reducing agent in the presence of benzoic acid in DMSO/DMF (60:40 v/v) at 100 °C for 10 min gave (R,S)-[11C]isoproterenol in an 87% radio-high performance liquid chromatography (HPLC) analytical yield. HPLC separation using a strong cation exchange column, followed by pharmaceutical formulation in the presence of d/l-tartaric acid, afforded (R,S)-[11C]isoproterenol with a total radioactivity of 2.0 ±â€¯0.2 GBq, a decay-corrected radiochemical yield of 19 ±â€¯2%, chemical and radiochemical purities of 71% and >99%, respectively, and a molar activity of 100 ±â€¯13 GBq/µmol (n = 3). The overall synthesis time from the end of the bombardment to pharmaceutical formulation was 48 min. A preliminary preclinical PET study in a rat demonstrated the potential of the radioligand for the evaluation of the penetration of (R,S)-isoproterenol in human brain.

Synthesis of antisense oligonucleotides containing acyclic alkynyl nucleoside analogs and their biophysical and biological properties.

The synthesis of oligonucleotide (ON) analogs, which can be used as antisense molecules, has recently gained much attention. Here, we report the synthesis and properties of an ON analog containing acyclic thymidine and cytidine analogs with a 4-pentyl-1,2-diol instead of the d-ribofuranose moiety. The incorporation of these analogs into the ON improved its nuclease resistance to 3'-exonucleases. Furthermore, it was found that the incorporation of the acyclic thymidine analog into a DNA/RNA duplex accelerates the RNA cleavage of a DNA/RNA duplex by Escherichia coli RNase H.

Incorporation of an acyclic alkynyl nucleoside analog into siRNA improves silencing activity and nuclease resistance.

In order to improve the silencing activity and nuclease resistance of small interfering RNA (siRNA), we designed and synthesized an acyclic thymidine analog containing 4-pentyne-1,2-diol instead of d-ribofuranose. The incorporation of this analog into siRNAs at specific positions in the strands was found to enhance the silencing activity of siRNAs and to increase the resistance of the siRNA to hydrolytic degradation by a 3' exonuclease.

Autoactivation of pancreatic trypsinogen is controlled by carbohydrate-specific interaction.

Activation of bovine pancreatic trypsinogen (BPTG) by trypsin (BPT) was found to be inhibited by d GalN/GalNAc at pH 5.5, the pH of secretory granules in the pancreas. Binding studies with biotinylated sugar-polymers indicated that BPTG and BPT bind to α-GalNAc, α-Man, and α-Gal better at pH 5.5 than at pH 7.5. Ultraviolet-difference spectra indicated that BPTG binding to α-GalNAc differs substantially from BPTG binding to other sugars. The N-α-benzoyl-d,l-arginine-p-nitroanilide hydrochloride-hydrolyzing activity of BPT was only slightly affected by these sugars. The results indicate that the binding of GalNAc - containing glycoconjugates protects BPTG from autoactivation, and this may be a self-defense mechanism against intrapancreatic activation.

An automated new technique for scoring the in vivo micronucleus assay with image analysis.

The mammalian erythrocyte micronucleus assay is frequently used to assess chemical-induced damage to the chromosomes or the mitotic apparatus of erythroblasts. Because quantitative analysis of micronuclei by microscopy is time consuming and laborious, several automatic scoring methodologies with image analysis have been reported. However, there have been cases in which it was difficult to examine the proportion of polychromatic erythrocytes (PCEs) among total erythrocytes as an index for bone marrow (BM) toxicity, and sample slide preparation has proven to be laborious with existing automatic methods. We developed an automatic scoring system with image analysis for the rodent erythrocyte micronucleus assay using 12-well plates employing high-content screening analyser. In our method, micronucleated PCEs (MNPCEs), PCEs and erythrocytes were identified from three kinds of images: bright field image, fluorescence image with Hoechst 33342, and fluorescence image with propidium iodide. The frequencies of MNPCEs and PCEs were subsequently calculated. A comparison of automatic and manual scoring was carried out using BM and peripheral blood (PB) obtained from mice treated with stepwise doses of mitomycin C. The scores obtained by automatic analysis corresponded to those obtained by manual scoring; the frequencies of MNPCEs in BM and PB obtained by automatic scoring were 132 and 113%, respectively, of those obtained by manual scoring, and the corresponding frequencies of PCEs were 95 and 120%, respectively. Furthermore, we performed five repeats of the examinations of mouse BM and PB treated with mitomycin C or vinblastine sulphate and showed that automatic scoring was equivalent to manual scoring in reproducibility. Meanwhile, the scoring data obtained by manual scoring tended to vary among observers. These results suggest that our automatic scoring system with image analysis is superior to manual microscopy scoring in terms of speed and objectivity, comparable in reproducibility and useful for the in vivo micronucleus assay.

Evaluation of high-throughput screening for in vitro micronucleus test using fluorescence-based cell imaging.

The in vitro micronucleus (MN) test is an important component of genotoxicity screening and is used as an alternative to the in vitro chromosome aberration (CA) test. As the MN assay is more practical and simpler to use than the CA test, it is being applied as a high-throughput screening (HTS) assay. Therefore, we conducted a validation study of the MN test employing a confocal imaging plate reader, the IN Cell Analyzer 1000. We evaluated 30 chemicals, including clastogens and aneugens, using Chinese hamster lung cells (CHL/IU) seeded in 96-well microplates. The microplates were stained with Hoechst 33342 and CellMask Red for automated analysis, and MN were identified and counted automatically in fluorescence images. The MN test results for 30 chemicals obtained with this image analysis system, using the IN Cell Analyzer, were highly consistent with reference data for the in vitro MN test and CA test data obtained by microscopic analysis. In conclusion, this HTS assay for detecting MN offers high efficiency and accuracy in the early stages of chemical development.