Background: The P2X7 receptor represents a novel molecular target for imaging neuroinflammation via PET. GSK1482160, a potent P2X7 receptor antagonist, has high receptor affinity, blood-brain barrier penetration, and has been recently radiolabel (¹¹C-GSK1482160). Therefore, we report the initial physical and biological characterization of this novel ligand. Methods: ¹¹C-GSK1482160 synthesis was according to published methods. Cell density studies were performed in human embryonic kidney cell lines expressing human P2X7 receptors (HEK293-hP2X7R) and analyzed by western blot analysis, immuno-fluorescence assay (IFA), and radiologic-immunohistochemistry (RIHC) using P2X7R polyclonal antibodies. Receptor density (Bmax) and binding potential (Bp) were determined by saturation and association/disassociation kinetics, respectively. Peak immune response to lipopolysaccharide (LPS) stimuli in mice were determined in time course studies and analyzed via Iba1 and P2X7R western blot and Iba1 IHC. Whole animal biodistribution was performed in untreated mice or LPS at 15, 30, and 60min post radiotracer administration. Dynamic in vivo PET/CT was performed in mice administered saline, LPS, or LPS+blocking at 72 hr, and brain regions modeled using a 2 compartment 5 parameter tracer kinetic model. Results: P2X7R linearly changed with concentrations and/or cell numbers. Using high specific activity ¹¹C-GSK1482160, Bmax and Kd were 1.15±0.12nM and 3.03±0.10 pmol/mg in HEK293-hP2X7R membranes, respectively. Determination of kon, koff, and Bp in HEK293-hP2X7R cells were 0.2312±0.01542 min-1.nM-1, 0.2547±0.0155 min-1, 1.0277±0.207, respectively. Whole brain Iba1 expression in LPS stimulated mice peaked by 72 hr. IHC and western blot analysis of P2X7R for saline and LPS brain sections showed a 1.8 and 1.7 fold increase in signal enhancement at 72 hr, respectively. Biodistribution of ¹¹C-GSK1482160 in untreated and LPS treated mice at 72 hr were statistically significant across all tissues studied. In vivo dynamic ¹¹C-GSK1482160 PET/CT in mice administered saline, LPS, or LPS+blocking at 72 hr resulted in a 3.2 fold increase and 97% blocking by 30 min. Total volumes of distributions (V_T) for multiple cortical regions and the hippocampus showed statistically significant increases, and were blocked by excess authentic standard GSK1482160. Conclusion: The current study provides compelling data that supports ¹¹C-GSK1482160 is suitable as a novel radioligand targeting P2X7 receptors as a biomarker of neuroinflammation.

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, P2X₇receptor 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. Severalin vivostudies have demonstrated beneficial effects of the P2X₇receptor antagonists in animal model systems of neurodegenerative diseases. A number of specific and selective P2X₇receptor antagonists have been developed, but only few of them have shown efficient brain permeability. Finding potent and selective P2X₇receptor 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 P2X₇receptor function in neurodegenerative diseases, the pharmacological inhibition of the receptor, and PET radiopharmaceuticals which permit non-invasive monitoring of the P2X₇receptor contribution to neuroinflammation associated with neurodegeneration.

Hydrocephalus is a serious condition that impacts patients of all ages. The standards of care are surgical options to divert, or inhibit production of, cerebrospinal fluid; to date, there are no effective pharmaceutical treatments, to our knowledge. The causes vary widely, but one commonality of this condition is aberrations in salt and fluid balance. We have used a genetic model of hydrocephalus to show that ventriculomegaly can be alleviated by inhibition of the transient receptor potential vanilloid 4, a channel that is activated by changes in osmotic balance, temperature, pressure and inflammatory mediators. The TRPV4 antagonists do not appear to have adverse effects on the overall health of the WT or hydrocephalic animals.

The early events that shape the innate immune response to restrain pathogens during skin infections remain elusive. Methicillin-resistant Staphylococcus aureus (MRSA) infection engages phagocyte chemotaxis, abscess formation, and microbial clearance. Upon infection, neutrophils and monocytes find a gradient of chemoattractants that influence both phagocyte direction and microbial clearance. The bioactive lipid leukotriene B₄ (LTB₄) is quickly (seconds to minutes) produced by 5-lipoxygenase (5-LO) and signals through the G protein-coupled receptors LTB4R1 (BLT1) or BLT2 in phagocytes and structural cells. Although it is known that LTB₄ enhances antimicrobial effector functions in vitro, whether prompt LTB₄ production is required for bacterial clearance and development of an inflammatory milieu necessary for abscess formation to restrain pathogen dissemination is unknown. We found that LTB₄ is produced in areas near the abscess and BLT1 deficient mice are unable to form an abscess, elicit neutrophil chemotaxis, generation of neutrophil and monocyte chemokines, as well as reactive oxygen species-dependent bacterial clearance. We also found that an ointment containing LTB₄ synergizes with antibiotics to eliminate MRSA potently. Here, we uncovered a heretofore unknown role of macrophage-derived LTB₄ in orchestrating the chemoattractant gradient required for abscess formation, while amplifying antimicrobial effector functions. Staphylococcus aureus skin infection is orchestrated by resident and recruited immune cells. The signals that required for the control of S. aureus infection have been studied. However, most studies have focused on the actions of inflammatory mediators produced after hours post infection and the early events (seconds to minutes) that shape innate immune response is poorly understood. Here, we employed novel techniques, along with epistatic and gain of function experimental procedures, to identify critical components involved in the control of MRSA skin infection. Here, we unveiled the role of skin macrophages in the generation of the lipid mediator leukotriene B₄ (LTB₄) that culminates in optimal chemokine/cytokine production, abscess formation, and bacterial clearance. An ointment containing LTB₄ and antibiotics improved skin host defense and abscess formation and also enhanced bacterial clearance. These results suggest that LTB₄ could be used locally as an immunotherapeutic agent to strengthen/restore innate immune host activation during MRSA skin infections.

Raloxifene positively affects mechanical properties of the bone matrix in part through modification of skeletal‐bound water. The goal of this study was to determine if raloxifene‐induced alterations in skeletal hydration could be measured in vivo using ultra‐short echotime magnetic resonance imaging (UTE‐MRI). Twelve skeletally mature female beagle dogs (n = 6/group) were treated for 6 months with oral doses of saline vehicle (VEH, 1 mL/kg/d) or raloxifene (RAL, 0.5 mg/kg/d). After 6 months of treatment, all animals underwent in vivo UTE‐MRI of the proximal tibial cortical bone. UTE‐MRI signal intensity versus echotime curves were analyzed by fitting a double exponential to determine the short and long relaxation times of water with the bone (dependent estimations of bound and free water, respectively). Raloxifene‐treated animals had significantly higher bound water (+14%; p = 0.05) and lower free water (–20%) compared with vehicle‐treated animals. These data provide the first evidence that drug‐induced changes in skeletal hydration can be noninvasively assessed using UTE‐MRI. © 2015 American Society for Bone and Mineral Research. © 2015 American Society for Bone and Mineral Research.