The In Vivo Biological Fate of Protein Corona: A Comparative PET Study of the Fate of Soft and Hard Protein Corona in Healthy Animal Models.

Radiolabeling and nuclear imaging techniques are used to investigate the biodistribution patterns of the soft and hard protein corona around poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) after administration to healthy mice. Soft and hard protein coronas of 131I-labeled BSA or 131I-labeled serum are formed on PLGA NPs functionalized with either polyehtylenimine (PEI) or bovine serum albumin (BSA). The exchangeability of hard and soft corona is assessed in vitro by gamma counting exposing PLGA NPs with corona to non-labeled BSA, serum, or simulated body fluid. PEI PLGA NPs form larger and more stable coronas than BSA PLGA NPs. Soft coronas are more exchangeable than hard ones. The in vivo fate of PEI PLGA NPs coated with preformed 18F-labeled BSA hard and soft coronas is assessed by positron emission tomography (PET) following intravenous administration. While the soft corona shows a biodistribution similar to free 18F BSA with high activity in blood and kidney, the hard corona follows patterns characteristic of nanoparticles, accumulating in the lungs, liver, and spleen. These results show that in vivo fates of soft and hard corona are different, and that soft corona is more easily exchanged with proteins from the body, while hard corona is largely retained on the nanoparticle surface.

Synthesis and PET Imaging Biodistribution Studies of Radiolabeled Iododiflunisal, a Transthyretin Tetramer Stabilizer, Candidate Drug for Alzheimer's Disease.

The small-molecule iododiflunisal (IDIF) is a transthyretin (TTR) tetramer stabilizer and acts as a chaperone of the TTR-Amyloid beta interaction. Oral administration of IDIF improves Alzheimer's Disease (AD)-like pathology in mice, although the mechanism of action and pharmacokinetics remain unknown. Radiolabeling IDIF with positron or gamma emitters may aid in the in vivo evaluation of IDIF using non-invasive nuclear imaging techniques. In this work, we report an isotopic exchange reaction to obtain IDIF radiolabeled with 18F. [19F/18F]exchange reaction over IDIF in dimethyl sulfoxide at 160 °C resulted in the formation of [18F]IDIF in 7 ± 3% radiochemical yield in a 20 min reaction time, with a final radiochemical purity of >99%. Biodistribution studies after intravenous administration of [18F]IDIF in wild-type mice using positron emission tomography (PET) imaging showed capacity to cross the blood-brain barrier (ca. 1% of injected dose per gram of tissue in the brain at t > 10 min post administration), rapid accumulation in the liver, long circulation time, and progressive elimination via urine. Our results open opportunities for future studies in larger animal species or human subjects.

Positron Emission Tomography Studies of the Biodistribution, Translocation, and Fate of Poly Allyl Amine-Based Carriers for Sirna Delivery by Systemic and Intratumoral Administration.

Polyamine-based vectors offer many advantages for gene therapy, but they are hampered by a limited knowledge on their biological fate and efficacy for nucleic acid delivery. The 18 F radiolabeled siRNA is complexed with poly(allyl amine) hydrochloride (PAH), PEGylated PAH (PAHPEG ), or oleic acid-modified PAH (PAHOleic ) to form polyplexes, and injected them intravenously into healthy rodents. The biodistribution patterns obtained by positron emission tomography (PET) imaging vary according to the polymer used for complexation. Free siRNA is quickly eliminated through the bladder. PAH and oleic acid modify PAH polyplexes accumulate in the lungs and liver. No elimination through the bladder is observed for PAH and PAHOleic within 2 h after administration. PAHPEG polyplexes accumulate in kidneys and are eliminated through the bladder. Polyplexes prepared with 18 F-labeled oleic acid-modified PAH and non-labeled siRNA show similar biodistribution to those prepared with labeled siRNA, but with more accumulation in the lungs due to the presence of non-complexed polymer. Intravenous administration of PAHOleic polyplexes in tumor models results in a limited availability of siRNA. When PAHOleic polyplexes are administered intratumorally in tumor bearing rodents, ≈40% of the radioactivity is retained in the tumor after 180 min while free siRNA is completely eliminated.

PET Imaging of Self-Assembled 18 F-Labelled Pd2 L4 Metallacages for Anticancer Drug Delivery.

To advance the design of self-assembled metallosupramolecular architectures as new generation theranostic agents, the synthesis of 18 F-labelled [Pd2 L4 ]4+ metallacages is reported. Different spectroscopic and bio-analytical methods support the formation of the host-guest cage-cisplatin complex. The biodistribution profiles of one of the cages, alone or encapsulating cisplatin have been studied by PET/CT imaging in healthy mice in vivo, in combination to ICP-MS ex vivo.

Increased uptake of the P2X7 receptor radiotracer 18 F-JNJ-64413739 in the brain and peripheral organs according to the severity of status epilepticus in male mice.

OBJECTIVE: The P2X7 receptor (P2X7R) is an important contributor to neuroinflammation, responding to extracellularly released adenosine triphosphate. Expression of the P2X7R is increased in the brain in experimental and human epilepsy, and genetic or pharmacologic targeting of the receptor can reduce seizure frequency and severity in preclinical models. Experimentally induced seizures also increase levels of the P2X7R in blood. Here, we tested 18 F-JNJ-64413739, a positron emission tomography (PET) P2X7R antagonist, as a potential noninvasive biomarker of seizure-damage and epileptogenesis. METHODS: Status epilepticus was induced via an intra-amygdala microinjection of kainic acid. Static PET studies (30 min duration, initiated 30 min after tracer administration) were conducted 48 h after status epilepticus via an intravenous injection of 18 F-JNJ-64413739. PET images were coregistered with a brain magnetic resonance imaging atlas, tracer uptake was determined in the different brain regions and peripheral organs, and values were correlated to seizure severity during status epilepticus. 18 F-JNJ-64413739 was also applied to ex vivo human brain slices obtained following surgical resection for intractable temporal lobe epilepsy. RESULTS: P2X7R radiotracer uptake correlated strongly with seizure severity during status epilepticus in brain structures including the cerebellum and ipsi- and contralateral cortex, hippocampus, striatum, and thalamus. In addition, a correlation between radiotracer uptake and seizure severity was also evident in peripheral organs such as the heart and the liver. Finally, P2X7R radiotracer uptake was found elevated in brain sections from patients with temporal lobe epilepsy when compared to control. SIGNIFICANCE: Taken together, our data suggest that P2X7R-based PET imaging may help to identify seizure-induced neuropathology and temporal lobe epilepsy patients with increased P2X7R levels possibly benefitting from P2X7R-based treatments.

Binding of common organic UV-filters to the thyroid hormone transport protein transthyretin using in vitro and in silico studies: Potential implications in health.

Several anthropogenic contaminants have been identified as competing with the thyroid hormone thyroxine (T4) for binding to transport proteins as transthyretin (TTR). This binding can potentially create toxicity mechanisms posing a threat to human health. Many organic UV filters (UVFs) and paraben preservatives (PBs), widely used in personal care products, are chemicals of emerging concern due to their adverse effects as potential thyroid-disrupting compounds. Recently, organic UVFs have been found in paired maternal and fetal samples and PBs have been detected in placenta, which opens the possibility of the involvement of TTR in the transfer of these chemicals across physiological barriers. We aimed to investigate a discrete set of organic UVFs and PBs to identify novel TTR binders. The binding affinities of target UVFs towards TTR were evaluated using in vitro T4 competitive binding assays. The ligand-TTR affinities were determined by isothermal titration calorimetry (ITC) and compared with known TTR ligands. In parallel, computational studies were used to predict the 3-D structures of the binding modes of these chemicals to TTR. Some organic UVFs, compounds 2,2',4,4'-tetrahydroxybenzophenone (BP2, Kd = 0.43 µM); 2,4-dihydroxybenzophenone (BP1, Kd = 0.60 µM); 4,4'-dihydroxybenzophenone (4DHB, Kd = 0.83 µM), and 4-hydroxybenzophenone (4HB, Kd = 0.93 µM), were found to display a high affinity to TTR, being BP2 the strongest TTR binder (ΔH = -14.93 Kcal/mol). Finally, a correlation was found between the experimental ITC data and the TTR-ligand docking scores obtained by computational studies. The approach integrating in vitro assays and in silico methods constituted a useful tool to find TTR binders among common organic UVFs. Further studies on the involvement of the transporter protein TTR in assisting the transplacental transfer of these chemicals across physiological barriers and the long-term consequences of prenatal exposure to them should be pursued.

Novel Core-Shell Polyamine Phosphate Nanoparticles Self-Assembled from PEGylated Poly(allylamine hydrochloride) with Low Toxicity and Increased In Vivo Circulation Time.

An approach for reducing toxicity and enhancing therapeutic potential of supramolecular polyamine phosphate nanoparticles (PANs) through PEGylation of polyamines before their assembly into nanoparticles is presented here. It is shown that the number of polyethylene glycol (PEG) chains for polyamine largely influence physico-chemical properties of PANs and their biological endpoints. Poly(allylamine hydrochloride) (PAH) are functionalized through carbodiimide chemistry with three ratios of PEG molecules per PAH chain: 0.1, 1, and 10. PEGylated PAH is then assembled into PANs by exposing the polymer to phosphate buffer solution. PANs decrease size and surface charge with increasing PEG ratios as evidenced by dynamic light scattering and zeta potential measurements, with the ten PEG/PAH ratio PANs having practically zero charge. Small angle X-ray scattering (SAXS) proves that PEG chains form a shell around a polyamine core, which is responsible for the screening of positive charges. MTT experiments show that the screening of amine groups decreases nanoparticle toxicity, with the lowest toxicity for the 10 PEG/PAH ratio. Fluorescence correlation spectroscopy (FCS) proves less interaction with proteins for PEGylated PANs. Positron emission tomography (PET) imaging of 18 F labelled PANs shows longer circulation time in healthy mice for PEGylated PANs than non-PEGylated ones.

In Vivo Tracking of the Degradation of Mesoporous Silica through 89 Zr Radio-Labeled Core-Shell Nanoparticles.

While mesoporous silica nanoparticles (MSNs) are extensively studied as high-potential drug delivery platforms, the successful clinical translation of these nanocarriers strongly depends on their biodistribution, biodegradation, and elimination patterns in vivo. Here, a novel method is reported to follow the in vivo degradation of MSNs by tracking a radioactive label embedded in the silica structure. Core-shell silica nanoparticles (NPs) with a dense core and a mesoporous shell are labeled with low quantities of the positron emitter 89 Zr, either in the dense core or in the mesoporous shell. In vivo positron emission tomography imaging and ex vivo organ measurements reveal a remarkable difference in the 89 Zr biodistribution between the shell-labeled and the core-labeled NPs. Release of the radiotracer from shell-labeled NPs is used as a probe of the extent of silica dissolution, and a prompt release of the radioisotope is observed, with partial excretion already in the first 2 h post injection, and a slower accumulation in bones over time. On the other hand, when 89 Zr is embedded in the nanoparticle core, the biodistribution remains largely unchanged during the first 6 h. These findings indicate that MSNs have fast, hour-scale, degradation kinetics in vivo.

Therapeutic Efficacy of Novel Antimicrobial Peptide AA139-Nanomedicines in a Multidrug-Resistant Klebsiella pneumoniae Pneumonia-Septicemia Model in Rats.

Antimicrobial peptides (AMPs) have seen limited clinical use as antimicrobial agents, largely due to issues relating to toxicity, short biological half-life, and lack of efficacy against Gram-negative bacteria. However, the development of novel AMP-nanomedicines, i.e., AMPs entrapped in nanoparticles, has the potential to ameliorate these clinical problems. The authors investigated two novel nanomedicines based on AA139, an AMP currently in development for the treatment of multidrug-resistant Gram-negative infections. AA139 was entrapped in polymeric nanoparticles (PNPs) or lipid-core micelles (MCLs). The antimicrobial activity of AA139-PNP and AA139-MCL was determined in vitro The biodistribution and limiting doses of AA139-nanomedicines were determined in uninfected rats via endotracheal aerosolization. The early bacterial killing activity of the AA139-nanomedicines in infected lungs was assessed in a rat model of pneumonia-septicemia caused by extended-spectrum ß-lactamase-producing Klebsiella pneumoniae In this model, the therapeutic efficacy was determined by once-daily (q24h) administration over 10 days. Both AA139-nanomedicines showed equivalent in vitro antimicrobial activities (similar to free AA139). In uninfected rats, they exhibited longer residence times in the lungs than free AA139 (∼20% longer for AA139-PNP and ∼80% longer for AA139-MCL), as well as reduced toxicity, enabling a higher limiting dose. In rats with pneumonia-septicemia, both AA139-nanomedicines showed significantly improved therapeutic efficacy in terms of an extended rat survival time, although survival of all rats was not achieved. These results demonstrate potential advantages that can be achieved using AMP-nanomedicines. AA139-PNP and AA139-MCL may be promising novel therapeutic agents for the treatment of patients suffering from multidrug-resistant Gram-negative pneumonia-septicemia.

An Assay for Screening Potential Drug Candidates for Alzheimer's Disease That Act as Chaperones of the Transthyretin and Amyloid-ß Peptides Interaction.

The protein transthyretin (TTR) modulates amyloid-ß (Aß) peptides deposition and processing and this physiological effect is further enhanced by treatment with iododiflunisal (IDIF), a small-molecule compound (SMC) with TTR tetramer stabilization properties, which behaves as chaperone of the complex. This knowledge has prompted us to design and optimize a rapid and simple high-throughput assay that relies on the ability of test compounds to form ternary soluble complexes TTR/Aß/SMC that prevent Aß aggregation. The method uses the shorter Aß(12-28) sequence which is cheaper and simpler to use while retaining the aggregation properties of their parents Aß(1-40) and Aß(1-42). The test is carried out in 96-plate wells that are UV monitored for turbidity during 6 h. Given its reproducibility, we propose that this test can be a powerful tool for efficient screening of SMCs that act as chaperones of the TTR/Aß interaction that may led to potential AD therapies.

Undiscovered Roles for Transthyretin: From a Transporter Protein to a New Therapeutic Target for Alzheimer's Disease.

Transthyretin (TTR), an homotetrameric protein mainly synthesized by the liver and the choroid plexus, and secreted into the blood and the cerebrospinal fluid, respectively, has been specially acknowledged for its functions as a transporter protein of thyroxine and retinol (the latter through binding to the retinol-binding protein), in these fluids. Still, this protein has managed to stay in the spotlight as it has been assigned new and varied functions. In this review, we cover knowledge on novel TTR functions and the cellular pathways involved, spanning from neuroprotection to vascular events, while emphasizing its involvement in Alzheimer's disease (AD). We describe details of TTR as an amyloid binding protein and discuss its interaction with the amyloid Aß peptides, and the proposed mechanisms underlying TTR neuroprotection in AD. We also present the importance of translating advances in the knowledge of the TTR neuroprotective role into drug discovery strategies focused on TTR as a new target in AD therapeutics.

Repurposing Benzbromarone for Familial Amyloid Polyneuropathy: A New Transthyretin Tetramer Stabilizer.

Transthyretin (TTR) is a homotetrameric protein involved in human amyloidosis, including familial amyloid polyneuropathy (FAP). Discovering small-molecule stabilizers of the TTR tetramer is a therapeutic strategy for these diseases. Tafamidis, the only approved drug for FAP treatment, is not effective for all patients. Herein, we discovered that benzbromarone (BBM), a uricosuric drug, is an effective TTR stabilizer and inhibitor against TTR amyloid fibril formation. BBM rendered TTR more resistant to urea denaturation, similarly to iododiflunisal (IDIF), a very potent TTR stabilizer. BBM competes with thyroxine for binding in the TTR central channel, with an IC50 similar to IDIF and tafamidis. Results obtained by isothermal titration calorimetry (ITC) demonstrated that BBM binds TTR with an affinity similar to IDIF, tolcapone and tafamidis, confirming BBM as a potent binder of TTR. The crystal structure of the BBM-TTR complex shows two molecules binding deeply in the thyroxine binding channel, forming strong intermonomer hydrogen bonds and increasing the stability of the TTR tetramer. Finally, kinetic analysis of the ability of BBM to inhibit TTR fibrillogenesis at acidic pH and comparison with other stabilizers revealed that benzbromarone is a potent inhibitor of TTR amyloidogenesis, adding a new interesting scaffold for drug design of TTR stabilizers.

124 I Radiolabeling of a AuIII -NHC Complex for In Vivo Biodistribution Studies.

AuIII complexes with N-heterocyclic carbene (NHC) ligands have shown remarkable potential as anticancer agents, yet their fate in vivo has not been thoroughly examined and understood. Reported herein is the synthesis of new AuIII -NHC complexes by direct oxidation with radioactive [124 I]I2 as a valuable strategy to monitor the in vivo biodistribution of this class of compounds using positron emission tomography (PET). While in vitro analyses provide direct evidence for the importance of AuIII -to-AuI reduction to achieve full anticancer activity, in vivo studies reveal that a fraction of the AuIII -NHC prodrug is not immediately reduced after administration but able to reach the major organs before metabolic activation.

Immuno-PET Imaging and Pharmacokinetics of an Anti-CEA scFv-based Trimerbody and Its Monomeric Counterpart in Human Gastric Carcinoma-Bearing Mice.

Monoclonal antibodies (mAbs) are currently used as therapeutic agents in different types of cancer. However, mAbs and antibody fragments developed so far show suboptimal properties in terms of circulation time and tumor penetration/retention. Here, we report the radiolabeling, pharmacokinetic evaluation, and determination of tumor targeting capacity of the previously validated anti-CEA MFE23-scFv-based N-terminal trimerbody (MFE23N-trimerbody), and the results are compared to those obtained for the monomeric MFE23-scFv. Dissection and gamma-counting studies performed with the 131I-labeled protein scaffolds in normal mice showed slower blood clearance for the trimerbody, and accumulation in the kidneys, the spleen, and the liver for both species. These, together with a progressive uptake in the small intestine, confirm a combined elimination scheme with hepatobiliary and urinary excretion. Positron emission tomography studies performed in a xenograft mouse model of human gastric adenocarcinoma, generated by subcutaneous administration of CEA-positive human MKN45 cells, showed higher tumor accumulation and tumor-to-muscle (T/M) ratios for 124I-labeled MFE23N-trimerbody than for MFE23-scFv. Specific uptake was not detected with PET imaging in CEA negative xenografts as indicated by low T/M ratios. Our data suggest that engineered intermediate-sized trivalent antibody fragments could be promising candidates for targeted therapy and imaging of CEA-positive tumors.

Gold Nanoparticles as Boron Carriers for Boron Neutron Capture Therapy: Synthesis, Radiolabelling and In vivo Evaluation.

Background: Boron Neutron Capture Therapy (BNCT) is a binary approach to cancer therapy that requires accumulation of boron atoms preferentially in tumour cells. This can be achieved by using nanoparticles as boron carriers and taking advantage of the enhanced permeability and retention (EPR) effect. Here, we present the preparation and characterization of size and shape-tuned gold NPs (AuNPs) stabilised with polyethylene glycol (PEG) and functionalized with the boron-rich anion cobalt bis(dicarbollide), commonly known as COSAN. The resulting NPs were radiolabelled with 124I both at the core and the shell, and were evaluated in vivo in a mouse model of human fibrosarcoma (HT1080 cells) using positron emission tomography (PET). Methods: The thiolated COSAN derivatives for subsequent attachment to the gold surface were synthesized by reaction of COSAN with tetrahydropyran (THP) followed by ring opening using potassium thioacetate (KSAc). Iodination on one of the boron atoms of the cluster was also carried out to enable subsequent radiolabelling of the boron cage. AuNPs grafted with mPEG-SH (5 Kda) and thiolated COSAN were prepared by ligand displacement. Radiolabelling was carried out both at the shell (isotopic exchange) and at the core (anionic absorption) of the NPs using 124I to enable PET imaging. Results: Stable gold nanoparticles simultaneously functionalised with PEG and COSAN (PEG-AuNPs@[4]-) with hydrodynamic diameter of 37.8 ± 0.5 nm, core diameter of 19.2 ± 1.4 nm and ξ-potential of -18.0 ± 0.7 mV were obtained. The presence of the COSAN on the surface of the NPs was confirmed by Raman Spectroscopy and UV-Vis spectrophotometry. PEG-AuNPs@[4]- could be efficiently labelled with 124I both at the core and the shell. Biodistribution studies in a xenograft mouse model of human fibrosarcoma showed major accumulation in liver, lungs and spleen, and poor accumulation in the tumour. The dual labelling approach confirmed the in vivo stability of the PEG-AuNPs@[4]-. Conclusions: PEG stabilized, COSAN-functionalised AuNPs could be synthesized, radiolabelled and evaluated in vivo using PET. The low tumour accumulation in the animal model assayed points to the need of tuning the size and geometry of the gold core for future studies.

In vivo imaging of Α7 nicotinic receptors as a novel method to monitor neuroinflammation after cerebral ischemia.

In vivo positron emission tomography (PET) imaging of nicotinic acetylcholine receptors (nAChRs) is a promising tool for the imaging evaluation of neurologic and neurodegenerative diseases. However, the role of α7 nAChRs after brain diseases such as cerebral ischemia and its involvement in inflammatory reaction is still largely unknown. In vivo and ex vivo evaluation of α7 nAChRs expression after transient middle cerebral artery occlusion (MCAO) was carried out using PET imaging with [11 C]NS14492 and immunohistochemistry (IHC). Pharmacological activation of α7 receptors was evaluated with magnetic resonance imaging (MRI), [18 F]DPA-714 PET, IHC, real time polymerase chain reaction (qPCR) and neurofunctional studies. In the ischemic territory, [11 C]NS14492 signal and IHC showed an expression increase of α7 receptors in microglia and astrocytes after cerebral ischemia. The role played by α7 receptors on neuroinflammation was supported by the decrease of [18 F]DPA-714 binding in ischemic rats treated with the α7 agonist PHA 568487 at day 7 after MCAO. Moreover, compared with non-treated MCAO rats, PHA-treated ischemic rats showed a significant reduction of the cerebral infarct volumes and an improvement of the neurologic outcome. PHA treatment significantly reduced the expression of leukocyte infiltration molecules in MCAO rats and in endothelial cells after in vitro ischemia. Despite that, the activation of α7 nAChR had no influence to the blood brain barrier (BBB) permeability measured by MRI. Taken together, these results suggest that the nicotinic α7 nAChRs play a key role in the inflammatory reaction and the leukocyte recruitment following cerebral ischemia in rats.

Sustainable and Continuous Synthesis of Enantiopure l-Amino Acids by Using a Versatile Immobilised Multienzyme System.

The enzymatic synthesis of α-amino acids is a sustainable and efficient alternative to chemical processes, through which achieving enantiopure products is difficult. To more address this synthesis efficiently, a hierarchical architecture that irreversibly co-immobilises an amino acid dehydrogenase with polyethyleneimine on porous agarose beads has been designed and fabricated. The cationic polymer acts as an irreversible anchoring layer for the formate dehydrogenase. In this architecture, the two enzymes and polymer colocalise across the whole microstructure of the porous carrier. This multifunctional heterogeneous biocatalyst was kinetically characterised and applied to the enantioselective synthesis of a variety of canonical and noncanonical α-amino acids in both discontinuous (batch) and continuous modes. The co-immobilised bienzymatic system conserves more than 50 % of its initial effectiveness after five batch cycles and 8 days of continuous operation. Additionally, the environmental impact of this process has been semiquantitatively calculated and compared with the state of the art.

Biocatalysis in radiochemistry: Enzymatic incorporation of PET radionuclides into molecules of biomedical interest.

Biocatalysis is emerging as a new approach to radiolabel biologically active molecules with short half-lived positron emitters for their use in positron emission tomography. Despite the golden era of biocatalysis in radiochemistry occurred in the 70s and 80s, advances in enzyme engineering during the last decade have significantly enhanced the toolbox of enzymes available for chemical reactions, which may find application in the context of radiochemistry. In the present review, we intend to give an overview of the biocatalytic approaches that have been reported during the last 4 decades for the synthesis of PET radiotracers using nitrogen-13, carbon-11, and fluorine-18.

In vivo PET imaging of the α4ß2 nicotinic acetylcholine receptor as a marker for brain inflammation after cerebral ischemia.

PET imaging of nicotinic acetylcholine receptors (nAChRs) could become an effective tool for the diagnosis and therapy evaluation of neurologic diseases. Despite this, the role of nAChRs α4ß2 receptors after brain diseases such as cerebral ischemia and its involvement in inflammatory reaction is still largely unknown. To investigate this, we performed in parallel in vivo magnetic resonance imaging (MRI) and positron emission tomography (PET) with 2[(18)F]-fluoro-A85380 and [(11)C]PK11195 at 1, 3, 7, 14, 21, and 28 d after middle cerebral artery occlusion (MCAO) in rats. In the ischemic territory, PET with 2[(18)F]-fluoro-A85380 and [(11)C]PK11195 showed a progressive binding increase from days 3-7, followed by a progressive decrease from days 14-28 after cerebral ischemia onset. Ex vivo immunohistochemistry for the nicotinic α4ß2 receptor and the mitochondrial translocator protein (18 kDa) (TSPO) confirmed the PET findings and demonstrated the overexpression of α4ß2 receptors in both microglia/macrophages and astrocytes from days 7-28 after experimental ischemic stroke. Likewise, the role played by α4ß2 receptors on neuroinflammation was supported by the increase of [(11)C]PK11195 binding in ischemic rats treated with the α4ß2 antagonist dihydro-ß-erythroidine hydrobromide (DHBE) at day 7 after MCAO. Finally, both functional and behavioral testing showed major impaired outcome at day 1 after ischemia onset, followed by a recovery of the sensorimotor function and dexterity from days 21-28 after experimental stroke. Together, these results suggest that the nicotinic α4ß2 receptor could have a key role in the inflammatory reaction underlying cerebral ischemia in rats.

Pharmacokinetic investigation of sildenafil using positron emission tomography and determination of its effect on cerebrospinal fluid cGMP levels.

Sildenafil (Viagra) is a selective inhibitor of phosphodiesterase type 5 (PDE5), which degrades cyclic guanosine monophosphate to the linear nucleotide. Sildenafil is acutely used in erectile dysfunction and chronically in pulmonary hypertension. Evidence in the last decade shows that sildenafil may have potential as a therapeutic option for Alzheimer's disease or other neurodegenerative disorders. The purpose of this work was to explore whether sildenafil crosses the blood-brain barrier. Pharmacokinetic properties of sildenafil in rodents were investigated using (11) C-radiolabeling followed by in vivo positron emission tomography (PET) and ex vivo tissue dissection and gamma counting. PET results in rats suggest penetration into the central nervous system. Ex vivo data in perfused animals suggest that trapping of [(11) C]sildenafil within the cerebral vascular endothelium limits accumulation in the central nervous system parenchyma. Peroral sildenafil administration to Macaca fascicularis and subsequent chemical analysis of plasma and cerebrospinal fluid (CSF) using liquid chromatography coupled with tandem mass spectrometry showed that drug content in the CSF was high enough to achieve PDE5 inhibition, which was also demonstrated by the significant increases in CSF cyclic guanosine monophosphate levels. Central actions of sildenafil include both relaxation of the cerebral vasculature and inhibition of PDE5 in neurons and glia. This central action of sildenafil may underlie its efficacy in neuroprotection models, and may justify the continued search for a PDE5 ligand suitable for PET imaging. Sildenafil interacts with phosphodiesterase type 5 (PDE5) expressed in the endothelium and/or smooth muscle cells of brain vessels and also crosses the blood-brain barrier to interact with PDE5 expressed in brain cells. At therapeutic doses, the concentration of sildenafil in the cerebrospinal fluid (CSF) is high enough to inhibit PDE5 in the neural cells (neurons and glia). In turn, the concentration of cGMP likely increases in parenchymal cells and, as shown in this report, in the CSF. Read the Editorial Highlight for this article on page 220. Cover Image for this issue: doi: 10.1111/jnc.13302.