Age-Related Deficits in Binaural Hearing: Contribution of Peripheral and Central Effects.

Pure-tone audiograms often poorly predict elderly humans' ability to communicate in everyday complex acoustic scenes. Binaural processing is crucial for discriminating sound sources in such complex acoustic scenes. The compromised perception of communication signals presented above hearing threshold has been linked to both peripheral and central age-related changes in the auditory system. Investigating young and old Mongolian gerbils of both sexes, an established model for human hearing, we demonstrate age-related supra-threshold deficits in binaural hearing using behavioral, electrophysiological, anatomical, and imaging methods. Binaural processing ability was measured as the binaural masking level difference (BMLD), an established measure in human psychophysics. We tested gerbils behaviorally with "virtual headphones," recorded single-unit responses in the auditory midbrain and evaluated gross midbrain and cortical responses using positron emission tomography (PET) imaging. Furthermore, we obtained additional measures of auditory function based on auditory brainstem responses, auditory-nerve synapse counts, and evidence for central inhibitory processing revealed by PET. BMLD deteriorates already in middle-aged animals having normal audiometric thresholds and is even worse in old animals with hearing loss. The magnitude of auditory brainstem response measures related to auditory-nerve function and binaural processing in the auditory brainstem also deteriorate. Furthermore, central GABAergic inhibition is affected by age. Because the number of synapses in the apical turn of the inner ear was not reduced in middle-aged animals, we conclude that peripheral synaptopathy contributes little to binaural processing deficits. Exploratory analyses suggest increased hearing thresholds, altered binaural processing in the brainstem and changed central GABAergic inhibition as potential contributors.

Preparation and PET/CT imaging of implant directed 68Ga-labeled magnetic nanoporous silica nanoparticles.

BACKGROUND: Implant infections caused by biofilm forming bacteria are a major threat in orthopedic surgery. Delivering antibiotics directly to an implant affected by a bacterial biofilm via superparamagnetic nanoporous silica nanoparticles could present a promising approach. Nevertheless, short blood circulation half-life because of rapid interactions of nanoparticles with the host's immune system hinder them from being clinically used. The aim of this study was to determine the temporal in vivo resolution of magnetic nanoporous silica nanoparticle (MNPSNP) distribution and the effect of PEGylation and clodronate application using PET/CT imaging and gamma counting in an implant mouse model. METHODS: PEGylated and non-PEGylated MNPSNPs were radiolabeled with gallium-68 (68Ga), implementing the chelator tris(hydroxypyridinone). 36 mice were included in the study, 24 mice received a magnetic implant subcutaneously on the left and a titanium implant on the right hind leg. MNPSNP pharmacokinetics and implant accumulation was analyzed in dependence on PEGylation and additional clodronate application. Subsequently gamma counting was performed for further final analysis. RESULTS: The pharmacokinetics and biodistribution of all radiolabeled nanoparticles could clearly be visualized and followed by dynamic PET/CT imaging. Both variants of 68Ga-labeled MNPSNP accumulated mainly in liver and spleen. PEGylation of the nanoparticles already resulted in lower liver uptakes. Combination with macrophage depletion led to a highly significant effect whereas macrophage depletion alone could not reveal significant differences. Although MNPSNP accumulation around implants was low in comparison to the inner organs in PET/CT imaging, gamma counting displayed a significantly higher %I.D./g for the tissue surrounding the magnetic implants compared to the titanium control. Additional PEGylation and/or macrophage depletion revealed no significant differences regarding nanoparticle accumulation at the implantation site. CONCLUSION: Tracking of 68Ga-labeled nanoparticles in a mouse model in the first critical hours post-injection by PET/CT imaging provided a better understanding of MNPSNP distribution, elimination and accumulation. Although PEGylation increases circulation time, nanoparticle accumulation at the implantation site was still insufficient for infection treatment and additional efforts are needed to increase local accumulation.

99mTc-HMPAO SPECT imaging reveals brain hypoperfusion during status epilepticus.

Status epilepticus (SE) is a clinical emergency with high mortality. SE can trigger neuronal death or injury and alteration of neuronal networks resulting in long-term cognitive decline or epilepsy. Among the multiple factors contributing to this damage, imbalance between oxygen and glucose requirements and brain perfusion during SE has been proposed. Herein, we aimed to quantify by neuroimaging the spatiotemporal course of brain perfusion during and after lithium-pilocarpine-induced SE in rats. To this purpose, animals underwent 99mTc-HMPAO SPECT imaging at different time points during and after SE using a small animal SPECT/CT system. 99mTc-HMPAO regional uptake was normalized to the injected dose. In addition, voxel-based statistical parametric mapping was performed. SPECT imaging showed an increase of cortical perfusion before clinical seizure activity onset followed by regional hypo-perfusion starting with the first convulsive seizure and during SE. Twenty-four hours after SE, brain 99mTc-HMPAO uptake was widely decreased. Finally, chronic epileptic animals showed regionally decreased perfusion affecting hippocampus and cortical sub-regions. Despite elevated energy and oxygen requirements, brain hypo-perfusion is present during SE. Our results suggest that insufficient compensation of required blood flow might contribute to neuronal damage and neuroinflammation, and ultimately to chronic epilepsy generated by SE.

Proof-of-concept that network pharmacology is effective to modify development of acquired temporal lobe epilepsy.

Epilepsy is a complex network phenomenon that, as yet, cannot be prevented or cured. We recently proposed network-based approaches to prevent epileptogenesis. For proof of concept we combined two drugs (levetiracetam and topiramate) for which in silico analysis of drug-protein interaction networks indicated a synergistic effect on a large functional network of epilepsy-relevant proteins. Using the intrahippocampal kainate mouse model of temporal lobe epilepsy, the drug combination was administered during the latent period before onset of spontaneous recurrent seizures (SRS). When SRS were periodically recorded by video-EEG monitoring after termination of treatment, a significant decrease in incidence and frequency of SRS was determined, indicating antiepileptogenic efficacy. Such efficacy was not observed following single drug treatment. Furthermore, a combination of levetiracetam and phenobarbital, for which in silico analysis of drug-protein interaction networks did not indicate any significant drug-drug interaction, was not effective to modify development of epilepsy. Surprisingly, the promising antiepileptogenic effect of the levetiracetam/topiramate combination was obtained in the absence of any significant neuroprotective or anti-inflammatory effects as indicated by multimodal brain imaging and histopathology. High throughput RNA-sequencing (RNA-seq) of the ipsilateral hippocampus of mice treated with the levetiracetam/topiramate combination showed that several genes that have been linked previously to epileptogenesis, were significantly differentially expressed, providing interesting entry points for future mechanistic studies. Overall, we have discovered a novel combination treatment with promise for prevention of epilepsy.

Effective hematopoietic stem cell-based gene therapy in a murine model of hereditary pulmonary alveolar proteinosis.

Hereditary pulmonary alveolar proteinosis due to GM-CSF receptor deficiency (herPAP) constitutes a life-threatening lung disease characterized by alveolar deposition of surfactant protein secondary to defective alveolar macrophage function. As current therapeutic options are primarily symptomatic, we have explored the potential of hematopoietic stem cell-based gene therapy. Using Csf2rb-/- mice, a model closely reflecting the human herPAP disease phenotype, we here demonstrate robust pulmonary engraftment of an alveolar macrophage population following intravenous transplantation of lentivirally corrected hematopoietic stem and progenitor cells. Engraftment was associated with marked improvement of critical herPAP disease parameters, including bronchoalveolar fluid protein, cholesterol and cytokine levels, pulmonary density on computed tomography scans, pulmonary deposition of Periodic Acid-Schiff+ material as well as respiratory mechanics. These effects were stable for at least nine months. With respect to engraftment and alveolar macrophage differentiation kinetics, we demonstrate the rapid development of CD11c+/SiglecF+ cells in the lungs from a CD11c-/SiglecF+ progenitor population within four weeks after transplantation. Based on these data, we suggest hematopoietic stem cell-based gene therapy as an effective and cause-directed treatment approach for herPAP.

Ex vivo characterization of neuroinflammatory and neuroreceptor changes during epileptogenesis using candidate positron emission tomography biomarkers.

OBJECTIVE: Identification of patients at risk of developing epilepsy before the first spontaneous seizure may promote the development of preventive treatment providing opportunity to stop or slow down the disease. METHODS: As development of novel radiotracers and on-site setup of existing radiotracers is highly time-consuming and expensive, we used dual-centre in vitro autoradiography as an approach to characterize the potential of innovative radiotracers in the context of epilepsy development. Using brain slices from the same group of rats, we aimed to characterise the evolution of neuroinflammation and expression of inhibitory and excitatory neuroreceptors during epileptogenesis using translational positron emission tomography (PET) tracers; 18 F-flumazenil (18 F-FMZ; GABAA receptor), 18 F-FPEB (metabotropic glutamate receptor 5; mGluR5), 18 F-flutriciclamide (translocator protein; TSPO, microglia activation) and 18 F-deprenyl (monoamine oxidase B, astroglia activation). Autoradiography images from selected time points after pilocarpine-induced status epilepticus (SE; baseline, 24 and 48 hours, 5, 10 and 15 days and 6 and 12-14 weeks after SE) were normalized to a calibration curve, co-registered to an MRI-based 2D region-of-interest atlas, and activity concentration (Bq/mm2 ) was calculated. RESULTS: In epileptogenesis-associated brain regions, 18 F-FMZ and 18 F-FPEB showed an early decrease after SE. 18 F-FMZ decrease was maintained in the latent phase and further reduced in the chronic epileptic animals, while 18 F-FPEB signal recovered from day 10, reaching baseline levels in chronic epilepsy. 18 F-flutriciclamide showed an increase of activated microglia at 24 hours after SE, peaking at 5-15 days and decreasing during the chronic phase. On the other hand, 18 F-deprenyl autoradiography showed late astrogliosis, peaking in the chronic phase. SIGNIFICANCE: Autoradiography revealed different evolution of the selected targets during epileptogenesis. Our results suggest an advantage of combined imaging of inter-related targets like glutamate and GABAA receptors, or microglia and astrocyte activation, in order to identify important interactions, especially when using PET imaging for the evaluation of novel treatments.

Targeting Chemokine Receptor CXCR4 and Translocator Protein for Characterization of High-Risk Plaque in Carotid Stenosis Ex Vivo.

Background and Purpose- This pilot study aims to demonstrate the feasibility of targeting molecular characteristics of high-risk atherosclerotic plaque in symptomatic and asymptomatic carotid stenosis (CS), that is, upregulation of the translocator protein (TSPO) and the chemokine receptor type 4 (CXCR4), by means of molecular imaging. Methods- In a translational setting, specimens of carotid plaques of patients with symptomatic and asymptomatic CS obtained by carotid endarterectomy were analyzed for the presence of TSPO and CXCR4 by autoradiography, using the positron emission tomography tracers 18F-GE180 and 68Ga-Pentixafor and evaluated by histopathology. In addition, 68Ga-Pentixafor positron emission tomography/computed tomography was performed in a patient with high-grade CS. Results- Distinct patterns of upregulation of TSPO (18F-GE180 uptake) and CXCR4 (68Ga-Pentixafor uptake) were identified in carotid plaque by autoradiography. The spatial distribution was associated with specific histological hallmarks that are established features of high-risk plaque: TSPO upregulation correlated with activated macrophages infiltration, whereas CXCR4 upregulation also corresponded to areas of intraplaque hemorrhage. 68Ga-Pentixafor uptake was significantly higher in plaques of symptomatic compared with asymptomatic CS. Clinical positron emission tomography revealed marked 68Ga-Pentixafor uptake in carotid plaque of a patient with high-grade CS. Conclusions- Clinical imaging of molecular signatures of high-risk atherosclerotic plaque is feasible and may become a promising diagnostic tool for comprehensive characterization of carotid disease. This methodology provides a platform for future studies targeting carotid plaque.

Imaging of chemokine receptor CXCR4 expression in culprit and nonculprit coronary atherosclerotic plaque using motion-corrected [68Ga]pentixafor PET/CT.

PURPOSE: The chemokine receptor CXCR4 is a promising target for molecular imaging of CXCR4+ cell types, e.g. inflammatory cells, in cardiovascular diseases. We speculated that a specific CXCR4 ligand, [68Ga]pentixafor, along with novel techniques for motion correction, would facilitate the in vivo characterization of CXCR4 expression in small culprit and nonculprit coronary atherosclerotic lesions after acute myocardial infarction by motion-corrected targeted PET/CT. METHODS: CXCR4 expression was analysed ex vivo in separately obtained arterial wall specimens. [68Ga]Pentixafor PET/CT was performed in 37 patients after stent-based reperfusion for a first acute ST-segment elevation myocardial infarction. List-mode PET data were reconstructed to five different datasets using cardiac and/or respiratory gating. Guided by CT for localization, the PET signals of culprit and various groups of nonculprit coronary lesions were analysed and compared. RESULTS: Ex vivo, CXCR4 was upregulated in atherosclerotic lesions, and mainly colocalized with CD68+ inflammatory cells. In vivo, elevated CXCR4 expression was detected in culprit and nonculprit lesions, and the strongest CXCR4 PET signal (median SUVmax 1.96; interquartile range, IQR, 1.55-2.31) was observed in culprit coronary artery lesions. Stented nonculprit lesions (median SUVmax 1.45, IQR 1.23-1.88; P = 0.048) and hot spots in naive remote coronary segments (median SUVmax 1.34, IQR 1.23-1.74; P = 0.0005) showed significantly lower levels of CXCR4 expression. Dual cardiac/respiratory gating provided the strongest CXCR4 PET signal and the highest lesion detectability. CONCLUSION: We demonstrated the basic feasibility of motion-corrected targeted PET/CT imaging of CXCR4 expression in coronary artery lesions, which was triggered by vessel wall inflammation but also by stent-induced injury. This novel methodology may serve as a platform for future diagnostic and therapeutic clinical studies targeting the biology of coronary atherosclerotic plaque.

[18 F]GE180 positron emission tomographic imaging indicates a potential double-hit insult in the intrahippocampal kainate mouse model of temporal lobe epilepsy.

OBJECTIVE: Accumulating evidence suggests that brain inflammation, elicited by epileptogenic insults, is involved in epilepsy development. Noninvasive nuclear imaging of brain inflammation in animal models of epileptogenesis represents a diagnostic in vivo approach with potential for direct translation into the clinic. Here, we investigated up-regulation of the translocator protein (TSPO) indicative of microglial activation by serial [18 F]GE180 positron emission tomographic (PET) imaging in a mouse model of temporal lobe epilepsy. METHODS: As epileptogenic insult, a status epilepticus (SE) was induced in mice by intrahippocampal injection of kainate. Post-SE mice injected with kainate and sham-injected mice were subjected to [18 F]GE180 PET scans before SE and at 2 days, 5-7 days, 2 weeks, 3 weeks, 7 weeks, and 14 weeks postinsult. For data evaluation, brain regions ipsilateral and contralateral to the injection site were outlined by coregistration with a standard mouse brain atlas, and percentage of injected dose per cubic centimeter was calculated. In addition, a statistical parametric mapping analysis, comparing post-SE mice to baseline, sham mice to baseline, and post-SE to sham mice was performed. RESULTS: Following SE, elevations in [18 F]GE180 uptake were most prominent in the ipsilateral hippocampus, occurring between 2 days and at least 7 weeks after SE, with a peak at 5-7 days after SE. In the contralateral hippocampus and other epilepsy-associated brain regions, increased tracer uptake was observed with a similar time profile but to a lesser extent. Moderate enhancement of tracer uptake was also evident in mice after sham surgery. SIGNIFICANCE: TSPO in vivo imaging reliably detects brain inflammation during epileptogenesis. These inflammatory processes most prominently affect the hippocampus ipsilateral to the injection site. Inflammation induced by the traumatic insult associated with surgery synergistically contributes to total brain inflammation and may also contribute to epileptogenesis. The revealed time course of neuroinflammation will help to identify appropriate time points for anti-inflammatory, potentially antiepileptogenic treatment.

Isoflurane prevents acquired epilepsy in rat models of temporal lobe epilepsy.

OBJECTIVE: Acquired epilepsy is a devastating long-term risk of various brain insults, including trauma, stroke, infections, and status epilepticus (SE). There is no preventive treatment for patients at risk. Attributable to the complex alterations involved in epileptogenesis, it is likely that multitargeted approaches are required for epilepsy prevention. We report novel preclinical findings with isoflurane, which exerts various nonanesthetic effects that may be relevant for antiepileptogenesis. METHODS: The effects of isoflurane were investigated in two rat models of SE-induced epilepsy: intrahippocampal kainate and systemic administration of paraoxon. Isoflurane was either administered during (kainate) or after (paraoxon) induction of SE. Magnetic resonance imaging was used to assess blood-brain barrier (BBB) dysfunction. Positron emission tomography was used to visualize neuroinflammation. Long-term electrocorticographic recordings were used to monitor spontaneous recurrent seizures. Neuronal damage was assessed histologically. RESULTS: In the absence of isoflurane, spontaneous recurrent seizures were common in the majority of rats in both models. When isoflurane was administered during kainate injection, duration and severity of SE were not affected, but only few rats developed spontaneous recurrent seizures. A similar antiepileptogenic effect was found when paraoxon-treated rats were exposed to isoflurane after SE. Moreover, in the latter model, isoflurane prevented BBB dysfunction and neurodegeneration, whereas isoflurane reduced neuroinflammation in the kainate model. INTERPRETATION: Given that isoflurane is a widely used volatile anesthetic, and is used for inhalational long-term sedation in critically ill patients at risk to develop epilepsy, our findings hold a promising potential to be successfully translated into the clinic. Ann Neurol 2016;80:896-908.

Neuroinflammation imaging markers for epileptogenesis.

Epilepsy can be a devastating disorder. In addition to debilitating seizures, epilepsy can cause cognitive and emotional problems with reduced quality of life. Therefore, the major aim is to prevent the disorder in the first place: identify, detect, and reverse the processes responsible for its onset, and monitor and treat its progression. Epilepsy often occurs following a latent period of months to years (epileptogenesis) as a consequence of a brain insult, such as head trauma, stroke, or status epilepticus. Although this latent period clearly represents a therapeutic window, we are not able to stratify patients at risk for long-term epilepsy, which is prerequisite for preventative clinical trials. Moreover, because of the length of the latent period, an early biomarker for treatment response would be of high value. Finally, mechanistic biomarkers of epileptogenesis may provide more profound insight in the process of disease development.

Clinically relevant strategies for lowering cardiomyocyte glucose uptake for 18F-FDG imaging of myocardial inflammation in mice.

PURPOSE: Myocardial inflammation is an emerging target for novel therapies and thus for molecular imaging. Positron emission tomography (PET) with (18)F-fluorodeoxyglucose (FDG) has been employed, but requires an approach for suppression of cardiomyocyte uptake. We tested clinically viable strategies for their suitability in mouse models in order to optimize preclinical imaging protocols. METHODS: C57BL/6 mice (n = 56) underwent FDG PET under various conditions. In healthy animals, the effect of low-dose (5 units/kg) or high-dose (500 units/kg, 15 min prior) intravenous heparin, extended fasting (18 h) and the impact of conscious injection with limited, late application of isoflurane anaesthesia after 40 min of conscious uptake were examined in comparison to ketamine/xylazine anaesthesia. Conscious injection/uptake strategies were further evaluated at 3 days after permanent coronary artery occlusion. RESULTS: Under continuous isoflurane anaesthesia, neither heparin administration nor extended fasting significantly impacted myocardial (18)F-FDG accumulation. Injection with 40 min uptake in awake mice resulted in a marked reduction of global myocardial (18)F-FDG uptake compared to standard isoflurane anaesthesia (5.7 ± 1.1 %ID/g vs 30.2 ± 7.9 %ID/g, p < 0.01). Addition of heparin and fasting further reduced uptake compared to conscious injection alone (3.8 ± 1.5 %ID/g, p < 0.01) similar to ketamine/xylazine (2.4 ± 2.2 %ID/g, p < 0.001). In the inflammatory phase, 3 days after myocardial infarction, conscious injection/uptake with and without heparin/fasting identified a marked increase in myocardial (18)F-FDG accumulation that was similar to that observed under ketamine/xylazine. CONCLUSION: Continuous isoflurane anaesthesia obscures any suppressive effect of heparin or fasting on cardiomyocyte glucose utilization. Conscious injection of FDG in rodents significantly reduces cardiomyocyte uptake and enables further suppression by heparin and fasting, similar to clinical observations. In contrast to ketamine/xylazine, this represents a more physiological, translatable strategy for suppression of cardiomyocyte (18)F-FDG uptake when targeting myocardial inflammation.

Targeting post-infarct inflammation by PET imaging: comparison of (68)Ga-citrate and (68)Ga-DOTATATE with (18)F-FDG in a mouse model.

UNLABELLED: Imaging of inflammation early after myocardial infarction (MI) is a promising approach to the guidance of novel molecular interventions that support endogenous healing processes. (18)F-FDG PET has been used, but may be complicated by physiological myocyte uptake. We evaluated the potential of two alternative imaging targets: lactoferrin binding by (68)Ga-citrate and somatostatin receptor binding by (68)Ga-DOTATATE. METHODS: C57Bl/6 mice underwent permanent coronary artery ligation. Serial PET imaging was performed 3 - 7 days after MI using (68)Ga-citrate, (68)Ga-DOTATATE, or (18)F-FDG with ketamine/xylazine suppression of myocyte glucose uptake. Myocardial perfusion was evaluated by (13)N-ammonia PET and cardiac geometry by contrast-enhanced ECG-gated CT. RESULTS: Mice exhibited a perfusion defect of 30 - 40% (of the total left ventricle) with apical anterolateral wall akinesia and thinning on day 7 after MI. (18)F-FDG with ketamine/xylazine suppression demonstrated distinct uptake in the infarct region, as well as in the border zone and remote myocardium. The myocardial standardized uptake value in MI mice was significantly higher than in healthy mice under ketamine/xylazine anaesthesia (1.9 ± 0.4 vs. 1.0 ± 0.1). (68)Ga images exhibited high blood pool activity with no specific myocardial uptake up to 90 min after injection (tissue-to-blood contrast 0.9). (68)Ga-DOTATATE was rapidly cleared from the blood, but myocardial SUV was very low (0.10 ± 0.03). CONCLUSION: Neither (68)Ga nor (68)Ga-DOTATATE is a useful alternative to (18)F-FDG for PET imaging of myocardial inflammation after MI in mice. Among the three tested approaches, (18)F-FDG with ketamine/xylazine suppression of cardiomyocyte uptake remains the most practical imaging marker of post-infarct inflammation.

What does a picture tell? In vivo imaging of ABC transporter function.

Activity of ABC transporters in tumor tissue or at the blood­brain barrier is believed to be responsible for treatment failure of substrate drugs. As this mechanism will not be present in every single patient, diagnostic tools to study transporter function are urgently needed. Many efforts were made over the past years to improve in vivo quantification of ABC transporter function by molecular imaging techniques. This includes development of new positron emitting tracers, but also the evaluation of modified experimental protocols using already existing tracers. In addition to imaging of transporter function in healthy animals or volunteers, results from disease models or human patients are covered in this review.

Optimizing SUV Analysis: A Multicenter Study on Preclinical FDG-PET/CT Highlights the Impact of Standardization.

PURPOSE: Preclinical imaging, with translational potential, lacks a standardized method for defining volumes of interest (VOIs), impacting data reproducibility. The aim of this study was to determine the interobserver variability of VOI sizes and standard uptake values (SUVmean and SUVmax) of different organs using the same [18F]FDG-PET and PET/CT datasets analyzed by multiple observers. In addition, the effect of a standardized analysis approach was evaluated. PROCEDURES: In total, 12 observers (4 beginners and 8 experts) analyzed identical preclinical [18F]FDG-PET-only and PET/CT datasets according to their local default image analysis protocols for multiple organs. Furthermore, a standardized protocol was defined, including detailed information on the respective VOI size and position for multiple organs, and all observers reanalyzed the PET/CT datasets following this protocol. RESULTS: Without standardization, significant differences in the SUVmean and SUVmax were found among the observers. Coregistering CT images with PET images improved the comparability to a limited extent. The introduction of a standardized protocol that details the VOI size and position for multiple organs reduced interobserver variability and enhanced comparability. CONCLUSIONS: The protocol offered clear guidelines and was particularly beneficial for beginners, resulting in improved comparability of SUVmean and SUVmax values for various organs. The study suggested that incorporating an additional VOI template could further enhance the comparability of the findings in preclinical imaging analyses.

Tariquidar and elacridar are dose-dependently transported by P-glycoprotein and Bcrp at the blood-brain barrier: a small-animal positron emission tomography and in vitro study.

Elacridar (ELC) and tariquidar (TQD) are generally thought to be nontransported inhibitors of P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP), but recent data indicate that they may also be substrates of these multidrug transporters (MDTs). The present study was designed to investigate potential transport of ELC and TQD by MDTs at the blood-brain barrier at tracer doses as used in positron emission tomography (PET) studies. We performed PET scans with carbon-11-labeled ELC and TQD before and after MDT inhibition in wild-type and transporter-knockout mice as well as in in vitro transport assays in MDT-overexpressing cells. Brain entrance of [(11)C]ELC and [(11)C]TQD administered in nanomolar tracer doses was found to be limited by Pgp- and Bcrp1-mediated efflux at the mouse blood-brain barrier. At higher, MDT-inhibitory doses, i.e., 15 mg/kg for TQD and 5 mg/kg for ELC, brain activity uptake of [(11)C]ELC at 25 minutes after tracer injection was 5.8 ± 0.3, 2.1 ± 0.2, and 7.5 ± 1.0-fold higher in wild-type, Mdr1a/b((-/-),()) and Bcrp1((-/-)) mice, respectively, but remained unchanged in Mdr1a/b((-/-))Bcrp1((-/-)) mice. Activity uptake of [(11)C]TQD was 2.8 ± 0.2 and 6.8 ± 0.4-fold higher in wild-type and Bcrp1((-/-)) mice, but remained unchanged in Mdr1a/b((-/-)) and Mdr1a/b((-/-))Bcrp1((-/-)) mice. Consistent with the in vivo findings, in vitro uptake assays in Pgp- and Bcrp1-overexpressing cell lines confirmed low intracellular accumulation of ELC and TQD at nanomolar concentrations and increased uptake at micromolar concentrations. As this study shows that microdoses can behave pharmacokinetically differently from MDT-inhibitory doses if a compound interacts with MDTs, conclusions from microdose studies should be drawn carefully.

Is switching from brand name to generic formulations of phenobarbital associated with loss of antiepileptic efficacy?: a pharmacokinetic study with two oral formulations (Luminal(®) vet, Phenoleptil(®)) in dogs.

BACKGROUND: In human medicine, adverse outcomes associated with switching between bioequivalent brand name and generic antiepileptic drug products is a subject of concern among clinicians. In veterinary medicine, epilepsy in dogs is usually treated with phenobarbital, either with the standard brand name formulation Luminal(®) or the veterinary products Luminal(®) vet and the generic formulation Phenoleptil(®). Luminal(®) and Luminal(®) vet are identical 100 mg tablet formulations, while Phenoleptil(®) is available in the form of 12.5 and 50 mg tablets. Following approval of Phenoleptil(®) for treatment of canine epilepsy, it was repeatedly reported by clinicians and dog owners that switching from Luminal(®) (human tablets) to Phenoleptil(®) in epileptic dogs, which were controlled by treatment with Luminal(®), induced recurrence of seizures. In the present study, we compared bioavailability of phenobarbital after single dose administration of Luminal(®) vet vs. Phenoleptil(®) with a crossover design in 8 healthy Beagle dogs. Both drugs were administered at a dose of 100 mg/dog, resulting in 8 mg/kg phenobarbital on average. RESULTS: Peak plasma concentrations (Cmax) following Luminal(®) vet vs. Phenoleptil(®) were about the same in most dogs (10.9 ± 0.92 vs. 10.5 ± 0.77 µg/ml), and only one dog showed noticeable lower concentrations after Phenoleptil(®) vs. Luminal(®) vet. Elimination half-life was about 50 h (50.3 ± 3.1 vs. 52.9 ± 2.8 h) without differences between the formulations. The relative bioavailability of the two products (Phenoleptil(®) vs. Luminal(®) vet.) was 0.98 ± 0.031, indicating that both formulations resulted in about the same bioavailability. CONCLUSIONS: Overall, the two formulations did not differ significantly with respect to pharmacokinetic parameters when mean group parameters were compared. Thus, the reasons for the anecdotal reports, if true, that switching from the brand to the generic formulation of phenobarbital may lead to recurrence of seizures are obviously not related to a generally lower bioavailability of the generic formulation, although single dogs may exhibit lower plasma levels after the generic formulation that could be clinically meaningful.

Refinement of systemic guinea pig deafening in hearing research: Sensorineural hearing loss induced by co-administration of kanamycin and furosemide via the leg veins.

Auditory disabilities have a large impact on the human population worldwide. Research into understanding and treating hearing disabilities has increased significantly in recent years. One of the most relevant animal species in this context is the guinea pig, which has to be deafened to study several of the hearing pathologies and develop novel therapies. Applying kanamycin subcutaneously and furosemide intravenously is a long-established method in hearing research, leading to permanent hearing loss without surgical intervention at the ear. The intravenous application of furosemide requires invasive surgery in the cervical area of the animals to expose the jugular vein, since a relatively large volume (1 ml per 500 g body weight) must be injected over a period of about 2.5 min. We have established a gentler alternative by applying the furosemide by puncture of the leg veins. For this, custom-made cannula-needle devices were built to allow the vein puncture and subsequent slow injection of the furosemide. This approach was tested in 11 guinea pigs through the foreleg via the cephalic antebrachial vein and through the hind leg via the saphenous vein. Frequency-specific hearing thresholds were measured before and after the procedure to verify normal hearing and successful deafening, respectively. The novel approach of systemic deafening was successfully implemented in 10 out of 11 animals. The Vena saphena was best suited to the application. Since the animals' condition, post leg vein application, was better in comparison to animals deafened by exposure of the Vena jugularis, the postulated refinement that reduced animal stress was deemed successful.

A novel positron emission tomography imaging protocol identifies seizure-induced regional overactivity of P-glycoprotein at the blood-brain barrier.

Approximately one-third of epilepsy patients are pharmacoresistant. Overexpression of P-glycoprotein and other multidrug transporters at the blood-brain barrier is thought to play an important role in drug-refractory epilepsy. Thus, quantification of regionally different P-glycoprotein activity in the brain in vivo is essential to identify P-glycoprotein overactivity as the relevant mechanism for drug resistance in an individual patient. Using the radiolabeled P-glycoprotein substrate (R)-[(11)C]verapamil and different doses of coadministered tariquidar, which is an inhibitor of P-glycoprotein, we evaluated whether small-animal positron emission tomography can quantify regional changes in transporter function in the rat brain at baseline and 48 h after a pilocarpine-induced status epilepticus. P-glycoprotein expression was additionally quantified by immunohistochemistry. To reveal putative seizure-induced changes in blood-brain barrier integrity, we performed gadolinium-enhanced magnetic resonance scans on a 7.0 tesla small-animal scanner. Before P-glycoprotein modulation, brain uptake of (R)-[(11)C]verapamil was low in all regions investigated in control and post-status epilepticus rats. After administration of 3 mg/kg tariquidar, which inhibits P-glycoprotein only partially, we observed increased regional differentiation in brain activity uptake in post-status epilepticus versus control rats, which diminished after maximal P-glycoprotein inhibition. Regional increases in the efflux rate constant k(2), but not in distribution volume V(T) or influx rate constant K(1), correlated significantly with increases in P-glycoprotein expression measured by immunohistochemistry. This imaging protocol proves to be suitable to detect seizure-induced regional changes in P-glycoprotein activity and is readily applicable to humans, with the aim to detect relevant mechanisms of pharmacoresistance in epilepsy in vivo.

A comparative small-animal PET evaluation of [11C]tariquidar, [11C]elacridar and (R)-[11C]verapamil for detection of P-glycoprotein-expressing murine breast cancer.

PURPOSE: One important mechanism for chemoresistance of tumours is overexpression of the adenosine triphosphate-binding cassette transporter P-glycoprotein (Pgp). Pgp reduces intracellular concentrations of chemotherapeutic drugs. The aim of this study was to compare the suitability of the radiolabelled Pgp inhibitors [(11)C]tariquidar and [(11)C]elacridar with the Pgp substrate radiotracer (R)-[(11)C]verapamil for discriminating tumours expressing low and high levels of Pgp using small-animal PET imaging in a murine breast cancer model. METHODS: Murine mammary carcinoma cells (EMT6) were continuously exposed to doxorubicin to generate a Pgp-overexpressing, doxorubicin-resistant cell line (EMT6AR1.0 cells). Both cell lines were subcutaneously injected into female athymic nude mice. One week after implantation, animals underwent PET scans with [(11)C]tariquidar (n = 7), [(11)C]elacridar (n = 6) and (R)-[(11)C]verapamil (n = 7), before and after administration of unlabelled tariquidar (15 mg/kg). Pgp expression in tumour grafts was evaluated by Western blotting. RESULTS: [(11)C]Tariquidar showed significantly higher retention in Pgp-overexpressing EMT6AR1.0 compared with EMT6 tumours: the mean ± SD areas under the time-activity curves in scan 1 from time 0 to 60 min (AUC(0-60)) were 38.8 ± 2.2 min and 25.0 ± 5.3 min (p = 0.016, Wilcoxon matched pairs test). [(11)C]Elacridar and (R)-[(11)C]verapamil were not able to discriminate Pgp expression in tumour models. Following administration of unlabelled tariquidar, both EMT6Ar1.0 and EMT6 tumours showed increases in uptake of [(11)C]tariquidar, [(11)C]elacridar and (R)-[(11)C]verapamil. CONCLUSION: Among the tested radiotracers, [(11)C]tariquidar performed best in discriminating tumours expressing high and low levels of Pgp. Therefore [(11)C]tariquidar merits further investigation as a PET tracer to assess Pgp expression levels in solid tumours.