P2X7 Receptors in Neurodegeneration: Potential Therapeutic Applications From Basic to Clinical Approaches

Purinergic receptors play important roles in central nervous system (CNS), where the bulk of these receptors are implicated in neuroinflammatory responses and regulation of cellular function of neurons, microglial and astrocytes. Within the P2X receptor family, P2X7 receptor is generally known for its inactivity in normal conditions and activation by moderately high concentrations (>100 μM) of extracellular adenosine 5'-triphosphate (ATP) released from injured cells as a result of brain injury or pathological conditions. Activation of P2X7R contributes to the activation and proliferation of microglia and directly contribute to neurodegeneration by provoking microglia-mediated neuronal death, glutamate-mediated excitotoxicity, and NLRP3 inflammasome activation that results in initiation, maturity and release of the pro-inflammatory cytokines and generation of reactive oxygen and nitrogen species. These components of the inflammatory response play important roles in many neural pathologies and neurodegeneration disorders. In CNS, expression of P2X7R on microglia, astrocytes, and oligodendrocytes are upregulated under neuroinflammatory conditions. Several in vivo studies have demonstrated beneficial effects of the P2X7 receptor antagonists in animal model systems of neurodegenerative diseases. A number of specific and selective P2X7 receptor antagonists have been developed, but only few of them have shown efficient brain permeability. Finding potent and selective P2X7 receptor inhibitors which are also CNS penetrable and display acceptable pharmacokinetics (PK) has presented challenges for both academic researchers and pharmaceutical companies. In this review, we discuss the role of P2X7 receptor function in neurodegenerative diseases, the pharmacological inhibition of the receptor, and PET radiopharmaceuticals which permit non-invasive monitoring of the P2X7 receptor contribution to neuroinflammation associated with neurodegeneration

Meeting report of the annual workshop on Principles and Techniques for Improving Preclinical to Clinical Translation in Alzheimer\u27s Disease research.

INTRODUCTION: The second annual 5-day workshop on Principles and Techniques for Improving Preclinical to Clinical Translation in Alzheimer\u27s Disease Research was held October 7-11, 2019, at The Jackson Laboratory in Bar Harbor, Maine, USA, and included didactic lectures and hands-on training. Participants represented a broad range of research across the Alzheimer\u27s disease (AD) field, and varied in career stages from trainees and early stage investigators to established faculty, with attendance from the United States, Europe, and Asia. METHODS: In line with the National Institutes of Health (NIH) initiative on rigor and reproducibility, the workshop aimed to address training gaps in preclinical drug screening by providing participants with the skills and knowledge required to perform pharmacokinetic, pharmacodynamics, and preclinical efficacy experiments. RESULTS: This innovative and comprehensive workshop provided training in fundamental skill sets for executing in vivo preclinical translational studies. DISCUSSION: The success of this workship is expected to translate into practical skills that will enable the goals of improving preclinical to clinical translational studies for AD. HIGHLIGHTS: Nearly all preclinical studies in animal models have failed to translate to successful efficacious medicines for Alzheimer\u27s disease (AD) patients. While a wide variety of potential causes of these failures have been proposed,deficiencies in knowledge and best practices for translational research are not being sufficiently addressed by common training practices. Here we present proceedings from an annual NIA-sponsored workshop focused specifically on preclinical testing paradigms for AD translational research in animal models aimed at enabling improved preclinical to clinical translation for AD

Purinergic Receptors of the Central Nervous System: Biology, PET Ligands, and Their Applications:

Purinergic receptors play important roles in central nervous system (CNS). These receptors are involved in cellular neuroinflammatory responses that regulate functions of neurons, microglial and astrocytes. Based on their endogenous ligands, purinergic receptors are classified into P1 or adenosine, P2X and P2Y receptors. During brain injury or under pathological conditions, rapid diffusion of extracellular adenosine triphosphate (ATP) or uridine triphosphate (UTP) from the damaged cells, promote microglial activation that result in the changes in expression of several of these receptors in the brain. Imaging of the purinergic receptors with selective Positron Emission Tomography (PET) radioligands has advanced our understanding of the functional roles of some of these receptors in healthy and diseased brains. In this review, we have accumulated a list of currently available PET radioligands of the purinergic receptors that are used to elucidate the receptor functions and participations in CNS disorders. We have also reviewed receptors lacking radiotracer, laying the foundation for future discoveries of novel PET radioligands to reveal these receptors roles in CNS disorders

Functional MRI Assessment of Renal Fibrosis in Rat Models

poster abstractIntroduction Renal fibrosis is a common consequence of chronic kidney diseases which affects a large population. Therefore, it is important to establish imaging based noninvasive biomarkers to monitor the progression or regression of renal fibrosis instead of biopsy. Magnetic resonance imaging (MRI) could provide both high spatial resolution and excellent tissue contrast for visualization of kidney morphology. Moreover, MRI is capable of assessing pseudo perfusion (Df) and perfusion fraction (Pf) with intra-voxel incoherent motion (IVIM) imaging (1), tissue oxygenation with T2* mapping (2), macromolecular composition with T1rho imaging (3) and kidney function (eGFR) with dynamic contrast enhanced (DCE) imaging (4). This study is aimed to evaluate the sensitivity of these MRI techniques to the renal fibrotic changes in a rat model. Methods A total of 4 rats were scanned at early (2-5 days) and late (25-35 days) time points after surgical intervention (unilateral ureteral obstruction to induce renal fibrosis) on a Siemens Tim Trio 3T scanner using an 80mm inner diameter 8-channel rat body coil (RAPID, USA) under a stable anesthetized condition. Axial images of 80mm FOV, 2mm slice thick and sub-millimeter in-place resolution were acquired for different functional MRI techniques with following parameters, respectively: IVIM with10 b-values of 0 - 750 s/mm2. T2*: with 10 TEs of 8 - 66 ms; T1rho: with 9 TSL times of 5 - 80 ms; DCE: with150 dynamic measurements at a temporal resolution of 1.01 s. before and after a 15s injection of 1.1 ml GD-DTPA through rat tail with a power injector. Functional data were processed and analyzed using custom MATLAB programs or analysis tools installed in the MRI console workstation. Results Figure 1 shows an anatomical image of the obstructed (R) and healthy (L) rat kidneys. Figures 2-4 show example T1rho map, IVIM Df map, and T2* map, respectively. Quantitative results based on ROI measurements are summarized in table 1. Changes consistent with the expected progression of fibrosis were observed in the obstructed kidney (R) while the healthy kidney (L) and muscle region remained stable. Figure 5 shows the DCE-MRI images at baseline as well as 45s, 95s and 240s after contrast infusion. The timing and intensity of signal changes are clearly different between two kidneys. Quantitative results of DCE-MRI data and comparison with PET study is reported in a separate abstract. Discussion High quality anatomical and functional images of rat kidney can be obtained on a clinical 3.0T MR scanner with dedicated small animal coils and optimized imaging techniques. The findings suggest that IVIM, T2*, T1rho and DCE can be used to assess and monitor different aspects of physiological changes in kidney fibrosis

Beta Cell Imaging—From Pre-Clinical Validation to First in Man Testing

There are presently no reliable ways to quantify human pancreatic beta cell mass (BCM) in vivo, which prevents an accurate understanding of the progressive beta cell loss in diabetes or following islet transplantation. Furthermore, the lack of beta cell imaging hampers the evaluation of the impact of new drugs aiming to prevent beta cell loss or to restore BCM in diabetes. We presently discuss the potential value of BCM determination as a cornerstone for individualized therapies in diabetes, describe the presently available probes for human BCM evaluation, and discuss our approach for the discovery of novel beta cell biomarkers, based on the determination of specific splice variants present in human beta cells. This has already led to the identification of DPP6 and FXYD2γa as two promising targets for human BCM imaging, and is followed by a discussion of potential safety issues, the role for radiochemistry in the improvement of BCM imaging, and concludes with an overview of the different steps from pre-clinical validation to a first-in-man trial for novel tracers

Synthesis and in vitro biological evaluation of new P2X7R radioligands [11C]halo-GSK1482160 analogs

The reference standards halo-GSK1482160 (F-, Br-, and I-) and their corresponding precursors desmethyl-halo-GSK1482160 (F-, Br-, and I-) were synthesized from (S)-1-methyl-5-oxopyrrolidine-2-carboxylic acid or (S)-5-oxopyrrolidine-2-carboxylic acid and 2-halo-3-(trifluoromethyl)benzylamine (F-, Br-, and I-) in one step with 45–93% yields. The target tracers [11C]halo-GSK1482160 (F-, Br-, and I-) were prepared from desmethyl-halo-GSK1482160 (F-, Br-, and I-) with [11C]CH3OTf under basic conditions (NaOH-Na2CO3, solid, w/w 1:2) through N-[11C]methylation and isolated by HPLC combined with SPE in 40–50% decay corrected radiochemical yield. The radiochemical purity was >99%, and the molar activity (AM) at end of bombardment (EOB) was 370–740 GBq/μmol. The potency of halo-GSK1482160 (F-, Br-, and I-) in comparison with GSK1482160 (Cl-) was determined by a radioligand competitive binding assay using [11C]GSK1482160, and the binding affinity Ki values for halo-GSK1482160 (F-, Br-, and I-) and GSK1482160 (Cl-) are 54.2, 2.5, 1.9 and 3.1 nM, respectively

Identification of elongated cilia and chiral malformation in TMEM67 mutant brains

poster abstractTransmembrane protein 67 (TMEM67) is encoded by one of four syndromic encephalocele genes. In humans a mutation in TMEM67 causes Meckel Gruber Syndrome, type 3 (MKS3) which is characterized by severe encephalocele and cystic kidneys and is usually fatal in the neonatal period. MKS3 is one of a spectrum of diseases known as ciliopathies because the proteins responsible for the disease are found in cells with the primary cilia. Primary cilia are a single, hair-like organelle that is found on the apical membrane of polarized cells and is thought to be involved in formation of left-right asymmetry during development as well as mechano- and chemo-reception. Here we characterize previously unreported details of cerebral phenotype in the Wistar polycystic kidney (Wpk) rats with a TMEM67 mutation. In choroid plexus (CP) epithelia of wild type animals, TMEM67 localizes to the plasma membrane and to a region close to the basal side of CP primary cilia. In a choroid plexus cell line that forms an epithelial sheet, the TMEM67 is found intracellularly but also localizes to the junctional complexes as evidenced by β catenin co-localization. Absence of normal TMEM67 leads to elongation of primary cilia in the ependymal cells lining the cerebral ventricles of the TMEM67-/- animals indicating that this protein is involved in the regulation of cilia length. Reduced aqueduct, bilateral dilatation with fusion of lateral ventricles, swelling of the hippocampus, and altered hindbrain histoarchitecture are noted in the TMEM67-/- rats. In the heterozygous animals mild asymmetric ventriculomegaly primarily on the left side is observed during early postnatal periods and continues into adulthood. These results suggest that TMEM67 is required for cilia length control and normal development of cerebral midline that maintains the symmetry of the left and right hemispheres. The Wpk rat model, orthologous to human MKS3, provides a unique model in which to study the development of both severe (TMEM67-/-) and mild (TMEM67+/-) hydrocephalus and other developmental abnormalities that are commonly found in human patients with ciliopathies

Evaluation of 11C-Acetate and 18 F-FDG PET/CT in mouse multidrug resistance gene-2 deficient mouse model of hepatocellular carcinoma

Background Hepatocellular carcinoma (HCC) remains a global health problem with unique diagnostic and therapeutic challenges, including difficulties in identifying the highest risk patients. Previous work from our lab has established the murine multidrug resistance-2 mouse (MDR2) model of HCC as a reasonable preclinical model that parallels the changes seen in human inflammatory associated HCC. The purpose of this study is to evaluate modalities of PET/CT in MDR2−/− mice in order to facilitate therapeutic translational studies from bench to bedside. Methods 18F-FDG and 11C-acetate PET/CT was performed on 12 m MDR2−/− mice (n = 3/tracer) with HCC and 12 m MDR2−/+ control mice (n = 3/tracer) without HCC. To compare PET/CT to biological markers of HCC and cellular function, serum alpha-fetoprotein (AFP), lysophosphatidic acid (LPA), cAMP and hepatic tumor necrosis factor α (TNFα) were quantified in 3-12 m MDR2−/− (n = 10) mice using commercially available ELISA analysis. To translate results in mice to patients 11C-acetate PET/CT was also performed in 8 patents suspected of HCC recurrence following treatment and currently on the liver transplant wait list. Results Hepatic18F-FDG metabolism was not significantly increased in MDR2−/− mice. In contrast, hepatic 11C-acetate metabolism was significantly elevated in MDR2−/− mice when compared to MDR2−/+ controls. Serum AFP and LPA levels increased in MDR2−/− mice contemporaneous with the emergence of HCC. This was accompanied by a significant decrease in serum cAMP levels and an increase in hepatic TNFα. In patients suspected of HCC recurrence there were 5 true positives, 2 true negatives and 1 suspected false 11C-acetate negative. Conclusions Hepatic 11C-acetate PET/CT tracks well with HCC in MDR2−/− mice and patients with underlying liver disease. Consequently 11C-acetate PET/CT is well suited to study 1) HCC emergence/progression in patients and 2) reduce animal numbers required to study new chemotherapeutics in murine models of HCC

Synthesis and preliminary biological evaluation of radiolabeled 5-BDBD analogs as new candidate PET radioligands for P2X4 receptor

P2X4 receptor has become an interesting molecular target for treatment and PET imaging of neuroinflammation and associated brain diseases such as Alzheimer’s disease. This study reports the first design, synthesis, radiolabeling and biological evaluation of new candidate PET P2X4 receptor radioligands using 5-BDBD, a specific P2X4 receptor antagonist, as a scaffold. 5-(3-Hydroxyphenyl)-1-[11C]methyl-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one (N-[11C]Me-5-BDBD analog, [11C]9) and 5-(3-Bromophenyl)-1-[11C]methyl-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one (N-[11C]Me-5-BDBD, [11C]8c) were prepared from their corresponding desmethylated precursors with [11C]CH3OTf through N-[11C]methylation and isolated by HPLC combined with SPE in 30–50% decay corrected radiochemical yields with 370–1110 GBq/µmol specific activity at EOB. 5-(3-[18F]Fluorophenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one ([18F]F-5-BDBD, [18F]5a) and 5-(3-(2-[18F]fluoroethoxy)phenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one ([18F]FE-5-BDBD, [18F]11) were prepared from their corresponding nitro- and tosylated precursors by nucleophilic substitution with K[18F]F/Kryptofix 2.2.2 and isolated by HPLC-SPE in 5–25% decay corrected radiochemical yields with 111–740 GBq/µmol specific activity at EOB. The preliminary biological evaluation of radiolabeled 5-BDBD analogs indicated these new radioligands have similar biological activity with their parent compound 5-BDBD

Synthesis and preliminary biological evaluation of [11C]methyl (2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucinate for the fractalkine receptor (CX3CR1)

The reference standard methyl (2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucinate (5) and its precursor 2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucine (6) were synthesized from 6-amino-2-mercaptopyrimidin-4-ol and BnBr with overall chemical yield 7% in five steps and 4% in six steps, respectively. The target tracer [11C]methyl (2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucinate ([11C]5) was prepared from the acid precursor with [11C]CH3OTf through O-[11C]methylation and isolated by HPLC combined with SPE in 40–50% radiochemical yield, based on [11C]CO2 and decay corrected to end of bombardment (EOB). The radiochemical purity was >99%, and the specific activity (SA) at EOB was 370–1110 GBq/μmol with a total synthesis time of ∼40-min from EOB. The radioligand depletion experiment of [11C]5 did not display specific binding to CX3CR1, and the competitive binding assay of ligand 5 found much lower CX3CR1 binding affinity