The P2X(7) receptor tracer [C-11]SMW139 as an in vivo marker of neuroinflammation in multiple sclerosis: a first-in man study

Purpose: The novel PET tracer [11C]SMW139 binds with high affinity to the P2X7 receptor, which is expressed on pro-inflammatory microglia. The purposes of this first in-man study were to characterise pharmacokinetics of [11C]SMW139 in patients with active relapsing remitting multiple sclerosis (RRMS) and healthy controls (HC) and to evaluate its potential to identify in vivo neuroinflammation in RRMS. / Methods: Five RRMS patients and 5 age-matched HC underwent 90-min dynamic [11C]SMW139 PET scans, with online continuous and manual arterial sampling to generate a metabolite-corrected arterial plasma input function. Tissue time activity curves were fitted to single- and two-tissue compartment models, and the model that provided the best fits was determined using the Akaike information criterion. / Results: The optimal model for describing [11C]SMW139 kinetics in both RRMS and HC was a reversible two-tissue compartment model with blood volume parameter and with the dissociation rate k4 fixed to the whole-brain value. Exploratory group level comparisons demonstrated an increased volume of distribution (VT) and binding potential (BPND) in RRMS compared with HC in normal appearing brain regions. BPND in MS lesions was decreased compared with non-lesional white matter, and a further decrease was observed in gadolinium-enhancing lesions. In contrast, increased VT was observed in enhancing lesions, possibly resulting from disruption of the blood-brain barrier in active MS lesions. In addition, there was a high correlation between parameters obtained from 60- to 90-min datasets, although analyses using 60-min data led to a slight underestimation in regional VT and BPND values. / Conclusions: This first in-man study demonstrated that uptake of [11C]SMW139 can be quantified with PET using BPND as a measure for specific binding in healthy controls and RRMS patients. Additional studies are warranted for further clinical evaluation of this novel neuroinflammation tracer

Preterm Delivery Disrupts the Developmental Program of the Cerebellum

A rapid growth in human cerebellar development occurs in the third trimester, which is impeded by preterm delivery. The goal of this study was to characterize the impact of preterm delivery on the developmental program of the human cerebellum. Still born infants, which meant that all development up to that age had taken place in-utero, were age paired with preterm delivery infants, who had survived in an ex-utero environment, which meant that their development had also taken place outside the uterus. The two groups were assessed on quantitative measures that included molecular markers of granule neuron, purkinje neuron and bergmann glia differentiation, as well as the expression of the sonic hedgehog signaling pathway, that is important for cerebellar growth. We report that premature birth and development in an ex-utero environment leads to a significant decrease in the thickness and an increase in the packing density of the cells within the external granular layer and the inner granular layer well, as a reduction in the density of bergmann glial fibres. In addition, this also leads to a reduced expression of sonic hedgehog in the purkinje layer. We conclude that the developmental program of the cerebellum is specifically modified by events that follow preterm delivery

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

Identification of new molecular targets for PET imaging of the microglial anti-inflammatory activation state

Microglia are potential targets for therapeutic intervention in neurological and neurodegenerative diseases affecting the central nervous system. In order to assess the efficacy of therapies aimed to reduce the tissue damaging activities of microglia and/or to promote the protective potential of these cells, suitable pre-clinical and clinical tools for the in vivo analysis of microglia activities and dynamics are required. The aim of this work was to identify new translational markers of the anti-inflammatory / protective state of microglia for the development of novel PET tracers. Methods: New translational markers of the anti-inflammatory/protective activation state of microglia were selected by bioinformatic approaches and were in vitro and ex vivo validated by qPCR and immunohistochemistry in rodent and human samples. Once a viable marker was identified, a novel PET tracer was developed. This tracer was subsequently confirmed by autoradiography experiments in murine and human brain tissues. Results: Here we provide evidence that P2RY12 expression increases in murine and human microglia following exposure to anti-inflammatory stimuli, and that its expression is modulated in the reparative phase of experimental and clinical stroke. We then synthesized a novel carbon-11 labeled tracer targeting P2RY12, showing increased binding in brain sections of mice treated with IL4, and low binding to brain sections of a murine stroke model and of a stroke patient. Conclusion: This study provides new translational targets for PET tracers for the anti-inflammatory/protective activation state of microglia and shows the potential of a rationale-based approach. It therefore paves the way for the development of novel non-invasive methodologies aimed to monitor the success of therapeutic approaches in various neurological diseases

Biomechanical Evaluation of Dual Plate Configurations for Femoral Shaft Fracture Fixation

Aim. This study aimed at comparing the mechanical properties of conventional and locking dual plates in adjacent and orthogonal orientations for the surgical fixation of transverse femoral shaft fractures. It also assessed the failure mechanics after dual adjacent and orthogonal locking plate removal. Methods. Thirty-two composite femurs were transversally osteotomized and randomly assigned for fixation with either dual locking or compression plates in an adjacent or orthogonal configuration. Sixteen specimens were preloaded axially to 20 N and single-leg stance loads were simulated. The remaining sixteen constructs were subjected to torsional loads of 10 Nm at a rate of 10 Nm/s in external and internal rotation of the femoral head in relation to the knee. Overall combined rotational stiffness was calculated. Eight different specimens with no osteotomy underwent the same experiments after dual locked plate removal and were tested to failure in combined eccentric axial and torsional modes. Data were statistically processed using a two-tailed t-test and one-way analysis of variance for the comparison of means between two or more groups, respectively. Results. Orthogonal constructs were statistically stiffer in axial loading compared to their adjacent counterparts in both conventional and locking configurations (p0.05). Conclusions. In both orthogonal and adjacent orientations, double locking plates provide higher stability than their dual conventional counterparts. Orthogonal dual plate configuration is more stable and biomechanically superior to dual adjacent plating for constructs fixed with either standard compression or locking plates

Nucleotide sequence variation in a small region of the Grapevine fleck virus replicase provides evidence for two sequence variants of the virus

A region encoding the putative RNA replicase domain of Grapevine fleck virus (GFkV) was amplified from infected grapevine samples using the reverse transcription-polymerase chain reaction (RT-PCR). Two different sizes of virus specific PCR products were consistently detected in different grapevine varieties using the same pair of primers. We selected four reference vines and carried out nucleotide sequence analysis. The size of the small product was 353 nucleotides (nt) and that of the large product was 416 nt. Database search showed that both products shared a high sequence homology with GFkV. Sequence variants producing the small size products were named GFkV₃₅₃, while those giving the large size products were named GFkV₄₁₆. The small size product shared a higher sequence homology with that of the published GFkV sequence from Italy, which had the same product size of 353 nt, while the large product had a 63 base insertion in that region. This insertion occurred within domain VI of the replicase gene. A survey was carried out for the presence of these two sequence variants in Australia, New Zealand, the USA, South Africa and a number of other countries. In Australia, of 3874 grapevine samples tested by the RT-PCR assay, 492 (12.7%) contained GFkV₃₅₃ and 145 (3.7%) contained GFkV₄₁₆, while only six (0.1%) contained both variants. The sequence homology between these two amplified products and the corresponding regions in related viruses of tymoviruses and marafiviruses is discussed

Synthesis and preclinical evaluation of [C-11]LR111 and [F-18]EW-7197 as PET tracers of the activin-receptor like kinase-5

The transforming growth factor beta (TGF beta) pathway plays a complex role in cancer biology, being involved in both tumour suppression as well as promotion. Overactive TGF beta signalling has been linked to multiple diseases, including cancer, pulmonary arterial hypertension, and fibrosis. One of the key meditators within this pathway is the TGF beta type I receptor, also termed activin receptor-like kinase 5 (ALK5). ALK5 expression level is a key determinant of TGF beta signalling intensity and duration, and perturbation has been linked to diseases. A validated ALK5 positron emission tomography (PET) tracer creates an opportunity, therefore, to study its role in human diseases. To develop ALK5 PET tracers, two small molecule ALK5 kinase inhibitors were selected as lead compounds, which were labelled with carbon-11 and fluorine-18, respectively. [C-11]LR111 was synthesized with a yield of 17 +/- 6%, a molar activity of 126 +/- 79 GBq. mol(-1) and a purity of > 95% (n = 44). [18F]EW-7197 was synthesized with a yield of 10 +/- 5%, a molar activity of 183 & PLUSMN; 126 GBq. mol(-1) and a purity of > 95% (n = 11). Metabolic stability was evaluated in vivo in mice, showing 39 +/- 2% of intact [11C]LR111 and 21 +/- 2% of intact [F-18]EW-7197 in blood plasma at 45 min p.i. In vitro binding experiments were conducted in breast cancer MDAMB-231 and lung cancer A431 cell lines. In addition, both tracers were used for PET imaging in MDA-MB-231 xenograft models. Selective uptake of [F-18]EW-7197 and [C-11]LR111 was observed in MDA-MB-231 cells, in the MDA-MB-231 tumour xenografts in vivo and in the autoradiograms. As [C-11]LR111 and [F-18]EW-7197 showed selectivity of binding to ALK5 in vivo and in vitro. Both tracers are thereby valuable tools for the detection of ALK5 activity.Cancer Signaling networks and Molecular Therapeutic

Synthesis and preclinical evaluation of [C-11]LR111 and [F-18]EW-7197 as PET tracers of the activin-receptor like kinase-5

The transforming growth factor beta (TGF beta) pathway plays a complex role in cancer biology, being involved in both tumour suppression as well as promotion. Overactive TGF beta signalling has been linked to multiple diseases, including cancer, pulmonary arterial hypertension, and fibrosis. One of the key meditators within this pathway is the TGF beta type I receptor, also termed activin receptor-like kinase 5 (ALK5). ALK5 expression level is a key determinant of TGF beta signalling intensity and duration, and perturbation has been linked to diseases. A validated ALK5 positron emission tomography (PET) tracer creates an opportunity, therefore, to study its role in human diseases. To develop ALK5 PET tracers, two small molecule ALK5 kinase inhibitors were selected as lead compounds, which were labelled with carbon-11 and fluorine-18, respectively. [C-11]LR111 was synthesized with a yield of 17 +/- 6%, a molar activity of 126 +/- 79 GBq. mol(-1) and a purity of > 95% (n = 44). [18F]EW-7197 was synthesized with a yield of 10 +/- 5%, a molar activity of 183 & PLUSMN; 126 GBq. mol(-1) and a purity of > 95% (n = 11). Metabolic stability was evaluated in vivo in mice, showing 39 +/- 2% of intact [11C]LR111 and 21 +/- 2% of intact [F-18]EW-7197 in blood plasma at 45 min p.i. In vitro binding experiments were conducted in breast cancer MDAMB-231 and lung cancer A431 cell lines. In addition, both tracers were used for PET imaging in MDA-MB-231 xenograft models. Selective uptake of [F-18]EW-7197 and [C-11]LR111 was observed in MDA-MB-231 cells, in the MDA-MB-231 tumour xenografts in vivo and in the autoradiograms. As [C-11]LR111 and [F-18]EW-7197 showed selectivity of binding to ALK5 in vivo and in vitro. Both tracers are thereby valuable tools for the detection of ALK5 activity