Blood-brain barrier P-glycoprotein function in healthy subjects and Alzheimer's disease patients: effect of polymorphisms in the ABCB1 gene

Background: P-glycoprotein is a blood-brain barrier efflux transporter involved in the clearance of amyloid-beta from the brain and, as such, might be involved in the pathogenesis of Alzheimer's disease. P-glycoprotein is encoded by the highly polymorphic ABCB1 gene. Single-nucleotide polymorphisms in the ABCB1 gene have been associated with altered P-glycoprotein expression and function. P-glycoprotein function at the blood-brain barrier can be quantified in vivo using the P-glycoprotein substrate tracer (R)-[11C]verapamil and positron emission tomography (PET). The purpose of this study was to assess the effects of C1236T, G2677T/A and C3435T single-nucleotide polymorphisms in ABCB1 on blood-brain barrier P-glycoprotein function in healthy subjects and patients with Alzheimer's disease. Methods: Thirty-two healthy subjects and seventeen patients with Alzheimer's disease underwent 60-min dynamic (R)-[11C]verapamil PET scans. The binding potential of (R)-[11C]verapamil was assessed using a previously validated constrained two-tissue plasma input compartment model and used as outcome measure. DNA was isolated from frozen blood samples and C1236T, G2677T/A and C3435T single-nucleotide polymorphisms were amplified by polymerase chain reaction. Results: In healthy controls, binding potential did not differ between subjects without and with one or more T present in C1236T, G2677T and C3435T. In contrast, patients with Alzheimer's disease with one or more T in C1236T, G2677T and C3435T had significantly higher binding potential values than patients without a T. In addition, there was a relationship between binding potential and T dose in C1236T and G2677T. Conclusions: In Alzheimer's disease patients, C1236T, G2677T/A and C3435T single-nucleotide polymorphisms may be related to changes in P-glycoprotein function at the blood-brain barrier. As such, genetic variations in ABCB1 might contribute to the progression of amyloid-beta deposition in the brain

Identifying advanced stage NSCLC patients who benefit from afatinib therapy using 18F-afatinib PET/CT imaging

Objectives: Non-small cell lung cancer (NSCLC) tumors harboring common (exon19del, L858R) and uncommon (e.g. G719X, L861Q) activating epidermal growth factor receptor (EGFR) mutations are best treated with EGFR tyrosine kinase inhibitors (TKI) such as the first-generation EGFR TKI erlotinib, second-generation afatinib or third-generation osimertinib. However, identifying these patients through biopsy is not always possible. Therefore, our aim was to evaluate whether 18F-afatinib PET/CT could identify patients with common and uncommon EGFR mutations. Furthermore, we evaluated the relation between tumor 18F-afatinib uptake and response to afatinib therapy.Materials and methods: 18F-afatinib PET/CT was performed in 12 patients: 6 EGFR wild type (WT), 3 EGFR common and 3 EGFR uncommon mutations. Tumor uptake of 18F-afatinib was quantified using TBR_WB60-90 (tumor-to-whole blood activity ratio 60-90 min post-injection) for each tumor. Response was quantified per lesion using percentage of change (PC): [(response measurement (RM)?baseline measurement (BM))/BM]?100. Statistical analyses were performed using t-tests, correlation plots and sensitivity/specificity analysis.Results: Twenty-one tumors were identified. Injected dose was 348 ? 31 MBq. Group differences were significant between WT versus EGFR (common and uncommon) activating mutations (p = 0.03). There was no significant difference between EGFR common versus uncommon mutations (p = 0.94). A TBR_WB60-90 cut-off value of 6 showed the best relationship with response with a sensitivity of 70 %, a specificity of 100 % and a positive predictive value of 100 %.Conclusion: 18F-afatinib uptake was higher in tumors with EGFR mutations (common and uncommon) compared to WT. Furthermore, a TBR_WB60-90 cut-off of 6 was found to best predict response to therapy. 18F-afatinib PET/ CT could provide a means to identify EGFR mutation positive patients who benefit from afatinib therapy.Pathogenesis and treatment of chronic pulmonary disease

Genome-wide CRISPR screens define determinants of epithelial-mesenchymal transition mediated immune evasion by pancreatic cancer cells

The genetic circuits that allow cancer cells to evade immune killing via epithelial mesenchymal plasticity remain poorly understood. Here, we showed that mesenchymal-like (Mes) KPC3 pancreatic cancer cells were more resistant to cytotoxic T lymphocyte (CTL)–mediated killing than the parental epithelial–like (Epi) cells and used parallel genome-wide CRISPR screens to assess the molecular underpinnings of this difference. Core CTL-evasion genes (such as IFN-γ pathway components) were clearly evident in both types. Moreover, we identified and validated multiple Mes-specific regulators of cytotoxicity, such as Egfr and Mfge8. Both genes were significantly higher expressed in Mes cancer cells, and their depletion sensitized Mes cancer cells to CTL-mediated killing. Notably, Mes cancer cells secreted more Mfge8 to inhibit proliferation of CD8+ T cells and production of IFN-γ and TNFα. Clinically, increased Egfr and Mfge8 expression was correlated with a worse prognosis. Thus, Mes cancer cells use Egfr-mediated intrinsic and Mfge8-mediated extrinsic mechanisms to facilitate immune escape from CD8+ T cells. Cancer Signaling networks and Molecular Therapeutic

Identification of the allosteric P2X7 receptor antagonist [11C]SMW139 as a PET tracer of microglial activation

The P2X7 receptor plays a significant role in microglial activation, and as a potential drug target, the P2X7 receptor is also an interesting target in positron emission tomography. The current study aimed at the development and evaluation of a potent tracer targeting the P2X7 receptor, to which end four adamantanyl benzamide analogues with high affinity for the human P2X7 receptor were labelled with carbon-11. All four analogues could be obtained in excellent radiochemical yield and high radiochemical purity and molar activity, and all analogues entered the rat brain. [11C]SMW139 showed the highest metabolic stability in rat plasma, and showed high binding to the hP2X7 receptor in vivo in a hP2X7 receptor overexpressing rat model. Although no significant difference in binding of [11C]SMW139 was observed between post mortem brain tissue of Alzheimer's disease patients and that of healthy controls in in vitro autoradiography experiments, [11C]SMW139 could be a promising tracer for P2X7 receptor imaging using positron emission tomography, due to high receptor binding in vivo in the hP2X7 receptor overexpressing rat model. However, further investigation of both P2X7 receptor expression and binding of [11C]SMW139 in other neurological diseases involving microglial activation is warranted

Evaluation of (R)-[11C]verapamil as PET tracer of P-glycoprotein function in the blood-brain barrier: kinetics and metabolism in the rat

There is evidence that P-glycoprotein (P-gp) in the blood-brain barrier (BBB) may be involved in the aetiology of neurological disorders. For quantification of P-gp function in vivo, (R)-[11C]verapamil can be used as a positron emission tomography (PET) tracer, provided that a mathematical model describing kinetics of uptake and clearance of verapamil is available. To develop and validate such a model, the kinetic profile and metabolism of (R)-[11C]verapamil have to be known. The aim of this study was to investigate the presence of labeled metabolites of [11C]verapamil in the plasma and (brain) tissue of Wistar rats. For this purpose, extraction and high-performance liquid chromatography (HPLC) methods were developed. The radioactive metabolites of (R)-[11C]verapamil in the liver were N-dealkylated compounds, O-demethylated compounds and a polar fraction formed from N-demethylation products of (R)-[11C]verapamil. Apart from this [11C] polar fraction, other radioactive metabolites of [11C]verapamil were not detected in the brain tissue. Thirty minutes after injection, unmetabolized (R)-[11C]verapamil accounted for 47% of radioactivity in the plasma and 69% in the brain. Sixty minutes after injection, unmetabolized (R)-[11C] verapamil was 27% and 48% in the plasma and the brain, respectively

Synthesis, structure activity relationship, radiolabeling and preclinical evaluation of high affinity ligands for the ion channel of the N-methyl-D-aspartate receptor as potential imaging probes for positron emission tomography

The N-methyl-d-aspartate receptor (NMDAr) is involved in many neurological and psychiatric disorders including Alzheimer's disease and schizophrenia. Currently, it is not possible to assess NMDAr availability in vivo. The purpose of this study was to develop a positron emission tomography (PET) ligand for the NMDAr ion channel. A series of di- and tri-N-substituted diarylguanidines was synthesized. In addition, in vitro binding affinity for the NMDAr ion channel in rat forebrain membrane fractions was assessed. Compounds 10, 11 and 32 were radiolabeled with either carbon-11 or fluorine-18. Ligands

Synthesis and preclinical evaluation of carbon-11 labelled N-((5-(4-fluoro-2-[C-11]methoxyphenyl) pyridin-3-yl) methyl) cyclopentanamine as a PET tracer for NR2B subunit-containing NMDA receptors

Introduction: The N-methyl-D-Aspartate (NMDA) receptor plays an important role in learning and memory. Overactivation is thought to play an important role in neurodegenerative disorders such as Alzheimer's disease. Currently, it is not possible to assess N-methyl-D-aspartate receptor (NMDAr) bio-availability in vivo. The purpose of this study was to develop a positron emission tomography (PET) ligand for the NR2B binding site of the NMDA receptor. Methods: N-((5-(4-fluoro-2-methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamine was radiolabelled with carbon-11 in the phenyl moiety. Biodistribution and blocking studies were carried out in anaesthetized mice and in non-anaesthetized rats. Results: N-((5-(4-fluoro-2-