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.

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.

Molecular imaging of fibroblast activation protein after myocardial infarction using the novel radiotracer [68Ga]MHLL1.

Background: Myocardial infarction (MI) evokes an organized remodeling process characterized by the activation and transdifferentiation of quiescent cardiac fibroblasts to generate a stable collagen rich scar. Early fibroblast activation may be amenable to targeted therapy, but is challenging to identify in vivo. We aimed to non-invasively image active fibrosis by targeting the fibroblast activation protein (FAP) expressed by activated (myo)fibroblasts, using a novel positron emission tomography (PET) radioligand [68Ga]MHLL1 after acute MI. Methods: One-step chemical synthesis and manual as well as module-based radiolabeling yielded [68Ga]MHLL1. Binding characteristics were evaluated in murine and human FAP-transfected cells, and stability tested in human serum. Biodistribution in healthy animals was interrogated by dynamic PET imaging, and metabolites were measured in blood and urine. The temporal pattern of FAP expression was determined by serial PET imaging at 7 d and 21 d after coronary artery ligation in mice as percent injected dose per gram (%ID/g). PET measurements were validated by ex vivo autoradiography and immunostaining for FAP and inflammatory macrophages. Results: [68Ga]MHLL1 displayed specific uptake in murine and human FAP-positive cells (p = 0.0208). In healthy mice the tracer exhibited favorable imaging characteristics, with low blood pool retention and dominantly renal clearance. At 7 d after coronary artery ligation, [68Ga]MHLL1 uptake was elevated in the infarct relative to the non-infarcted remote myocardium (1.3 ± 0.3 vs. 1.0 ± 0.2 %ID/g, p < 0.001) which persisted to 21 d after MI (1.3 ± 0.4 vs. 1.1 ± 0.4 %ID/g, p = 0.013). Excess unlabeled compound blocked tracer accumulation in both infarct and non-infarct remote myocardium regions (p < 0.001). Autoradiography and histology confirmed the regional uptake of [68Ga]MHLL1 in the infarct and especially border zone regions, as identified by Masson trichrome collagen staining. Immunostaining further delineated persistent FAP expression at 7 d and 21 d post-MI in the border zone, consistent with tracer distribution in vivo. Conclusion: The simplified synthesis of [68Ga]MHLL1 bears promise for non-invasive characterization of fibroblast activation protein early in remodeling after MI.

Optimization of Artificial Siderophores as 68Ga-Complexed PET Tracers for In Vivo Imaging of Bacterial Infections.

The diagnosis of bacterial infections at deep body sites benefits from noninvasive imaging of molecular probes that can be traced by positron emission tomography (PET). We specifically labeled bacteria by targeting their iron transport system with artificial siderophores. The cyclen-based probes contain different binding sites for iron and the PET nuclide gallium-68. A panel of 11 siderophores with different iron coordination numbers and geometries was synthesized in up to 8 steps, and candidates with the best siderophore potential were selected by a growth recovery assay. The probes [68Ga]7 and [68Ga]15 were found to be suitable for PET imaging based on their radiochemical yield, radiochemical purity, and complex stability in vitro and in vivo. Both showed significant uptake in mice infected with Escherichia coli and were able to discern infection from lipopolysaccharide-triggered, sterile inflammation. The study qualifies cyclen-based artificial siderophores as readily accessible scaffolds for the in vivo imaging of bacteria.

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.

Anesthesia and Preconditioning Induced Changes in Mouse Brain [18F] FDG Uptake and Kinetics.

PURPOSE: 2-Deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) has been widely used for imaging brain metabolism. Tracer injection in anesthetized animals is a prerequisite for performing dynamic positron emission tomography (PET) scanning. Since preconditioning, as well as anesthesia, has been described to potentially influence brain [18F] FDG levels, this study evaluated how these variables globally and regionally affect both [18F] FDG uptake and kinetics in murine brain. PROCEDURES: Sixty-minute dynamic [18F] FDG PET scans were performed in adult male C57BL/6 mice anesthetized with isoflurane [control (in 100 % O2), in medical air, in 100 % O2 + insulin pre-treatment, and in 100 % O2 after 18 h fasting], ketamine/xylazine, sevoflurane, and chloral hydrate. An additional group was scanned after awake uptake. Blood glucose levels were determined, and data was analyzed by comparing percent injected dose per cc tissue (%ID/cc) and glucose influx rate and metabolic rate (MRGlu) calculated by Patlak plot. RESULTS: Ketamine/xylazine and chloral hydrate anesthesia induced a lower whole-brain uptake of [18F] FDG (2.86 ± 0.67 %ID/cc, p < 0.001; 4.25 ± 0.28 %ID/cc, p = 0.0179, respectively) compared to isoflurane anesthesia (5.04 ± 0.19 %ID/cc). In addition, protocols affected differently distribution of [18F] FDG uptake in brain regions. Ketamine/xylazine reduced [18F] FDG influx rate in murine brain (0.0135 ± 0.0009 vs 0.0247 ± 0.0014 ml/g/min; p < 0.005) and chloral hydrate increased MRGlu (66.72 ± 3.75 vs 41.55 ± 3.06 µmol/min/100 ml; p < 0.01) compared to isoflurane. Insulin-pretreated animals showed a higher influx rate (0.0477 ± 0.0101 ml/min/g; p < 0.05) but a reduced MRGlu (21.92 ± 3.12 µmol/min/100 ml; p < 0.01). Blood glucose levels were negatively correlated to [18F] FDG uptake and influx rate, but positively correlated to MRGlu. CONCLUSIONS: Choice of anesthesia and pre-conditioning affect not only [18F] FDG uptake but also kinetics and regional distribution in the mouse brain. Both anesthesia and pre-conditioning should be carefully considered in the interpretation of [18F] FDG studies due to its great influence on the uptake and distribution of the tracer along the brain regions.

Immunohistochemical detection of chemokine receptor 4 expression in chronic osteomyelitis confirms specific uptake in 68Ga-Pentixafor-PET/CT.

Previous findings of our group showed the chemokine receptor CXCR4 as a suitable target in PET/CT-imaging of axial bone infections, early postoperative osteomyelitis and periprosthetic infections. The aim of this study was to verify specific uptake of 68Ga-Pentixafor in chronic osteomyelitis. METHODS: 29 consecutive patients who underwent 68Ga-Pentixafor-PET/CT with clinically suspected osteomyelitis were evaluated retrospectively. Bone tissues of 6 patients were available and evaluated by immunohistochemical staining for CXCR4 and autoradiography with 68Ga-Pentixafor. Staining was performed with an anti-CXCR4 antibody. In order to detect lymphocytic infiltration and CXCR4-expressing lymphocytes double immunofluorescence with an anti-CD3 and anti-CXCR4 antibody was performed. RESULTS: 68Ga-Pentixafor-PET/ CT was true positive in 16 and true negative in 13 patients. In available bone tissue samples, immunohistochemical staining of CXCR4 expression and autoradiography with 68Ga-Pentixafor was highly positive. Double immunofluorescence was able to detect CXCR4-expressing T-lymphocytes within all bone samples while a control sample of noninfected tibial bone was negative for CXCR4. CONCLUSION: 68Ga-Pentixafor-PET/CT specifically shows CXCR4-expressing immune cells in chronic osteomyelitis and is therefore a suitable method for imaging chronic infection of the skeleton.Der Chemokinrezeptor CXCR4 konnte in einer Pilotstudie unserer Arbeitsgruppe als geeignete Zielstruktur zur PET/CT-Bildgebung von frühen postoperativen und periprothetischen Osteomyelitiden sowie Osteomyelitiden im Stammskelett identifiziert werden. In dieser Studie haben wir untersucht, ob 68Ga-Pentixafor spezifisch CXCR4-exprimierende Entzündungszellen in einer chronischen Osteomyelitis darstellen kann. METHODEN: Es erfolgte eine retrospektive Auswertung von 29 Patienten mit klinischem Verdacht einer chronischen Osteomyelitis, die mittels 68Ga-Pentixafor-PET/CT untersucht wurden. Hiervon lagen uns in 6 Fällen Knochengewebe zur immunhistochemischen und autoradiographischen Evaluation vor. Die Immunhistochemie wurde mit einem anti-CXCR4 Antikörper durchgeführt. Des Weiteren wurden ein anti-CD3 und der anti-CXCR4-Antikörper zur Detektion CXCR4-exprimierender Lymphozyten am Ort der Entzündung mittels Doppel- Immunfluoreszenz verwendet. ERGEBNISSE: Die 68Ga-Pentixafor-PET/CT war bei 16 Patienten richtig positiv und bei 13 Patienten richtig negativ. Die Färbungen der verfügbaren Knochenpräparate waren sowohl in der Immunhistochemie als auch in der Autoradiographie deutlich positiv. In der Immunfluoreszenz konnten zudem CXCR4-exprimierende Lymphozyten am Ort der Entzündung in allen Proben nachgewiesen werden. Die Kontrolle eines Präparats einer nicht infizierten distalen Tibia zeigte dagegen keine CXCR4-oder CD3-Expression. FAZIT: Mit der 68Ga-Pentixafor-PET/CT können spezifisch CXCR4-exprimierende Lymphozyten am Ort der Entzündung nachgewiesen werden. Die 68Ga-Pentixafor-PET/CT stellt eine geeignete Methode in der Diagnostik chronischer Osteomyeltiden dar.

iPSC-Derived Macrophages Effectively Treat Pulmonary Alveolar Proteinosis in Csf2rb-Deficient Mice.

Induced pluripotent stem cell (iPSC)-derived hematopoietic cells represent a highly attractive source for cell and gene therapy. Given the longevity, plasticity, and self-renewal potential of distinct macrophage subpopulations, iPSC-derived macrophages (iPSC-Mφ) appear of particular interest in this context. We here evaluated the airway residence, plasticity, and therapeutic efficacy of iPSC-Mφ in a murine model of hereditary pulmonary alveolar proteinosis (herPAP). We demonstrate that single pulmonary macrophage transplantation (PMT) of 2.5-4 × 106 iPSC-Mφ yields efficient airway residence with conversion of iPSC-Mφ to an alveolar macrophage (AMφ) phenotype characterized by a distinct surface marker and gene expression profile within 2 months. Moreover, PMT significantly improves alveolar protein deposition and other critical herPAP disease parameters. Thus, our data indicate iPSC-Mφ as a source of functional macrophages displaying substantial plasticity and therapeutic potential that upon pulmonary transplantation will integrate into the lung microenvironment, adopt an AMφ phenotype and gene expression pattern, and profoundly ameliorate pulmonary disease phenotypes.

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.

Myocardial Inflammation Predicts Remodeling and Neuroinflammation After Myocardial Infarction.

BACKGROUND: The local inflammatory tissue response after acute myocardial infarction (MI) determines subsequent healing. Systemic interaction may induce neuroinflammation as a precursor to neurodegeneration. OBJECTIVES: This study sought to assess the influence of MI on cardiac and brain inflammation using noninvasive positron emission tomography (PET) of the heart-brain axis. METHODS: After coronary artery ligation or sham surgery, mice (n = 49) underwent serial whole-body PET imaging of the mitochondrial translocator protein (TSPO) as a marker of activated macrophages and microglia. Patients after acute MI (n = 3) were also compared to healthy controls (n = 9). RESULTS: Infarct mice exhibited elevated myocardial TSPO signal at 1 week versus sham (percent injected dose per gram: 8.0 ± 1.6 vs. 4.8 ± 0.9; p < 0.001), localized to activated CD68+ inflammatory cells in the infarct. Early TSPO signal predicted subsequent left ventricular remodeling at 8 weeks (rpartial = -0.687; p = 0.001). In parallel, brain TSPO signal was elevated at 1 week (1.7 ± 0.2 vs. 1.4 ± 0.2 for sham; p = 0.017), localized to activated microglia. After interval decline at 4 weeks, progressive heart failure precipitated a second wave of neuroinflammation (1.8 ± 0.2; p = 0.005). TSPO was concurrently up-regulated in remote cardiomyocytes at 8 weeks (8.8 ± 1.7, p < 0.001) without inflammatory cell infiltration, suggesting mitochondrial impairment. Angiotensin-converting enzyme inhibitor treatment lowered acute inflammation in the heart (p = 0.003) and brain (p = 0.06) and improved late cardiac function (p = 0.05). Patients also demonstrated elevation of cardiac TSPO signal in the infarct territory, paralleled by neuroinflammation versus controls. CONCLUSIONS: The brain is susceptible to acute MI and chronic heart failure. Immune activation may interconnect heart and brain dysfunction, a finding that provides a foundation for strategies to improve heart and brain outcomes.

[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.

Insulin supplementation attenuates cancer-induced cardiomyopathy and slows tumor disease progression.

Advanced cancer induces fundamental changes in metabolism and promotes cardiac atrophy and heart failure. We discovered systemic insulin deficiency in cachectic cancer patients. Similarly, mice with advanced B16F10 melanoma (B16F10-TM) or colon 26 carcinoma (C26-TM) displayed decreased systemic insulin associated with marked cardiac atrophy, metabolic impairment, and function. B16F10 and C26 tumors decrease systemic insulin via high glucose consumption, lowering pancreatic insulin production and producing insulin-degrading enzyme. As tumor cells consume glucose in an insulin-independent manner, they shift glucose away from cardiomyocytes. Since cardiomyocytes in both tumor models remained insulin responsive, low-dose insulin supplementation by subcutaneous implantation of insulin-releasing pellets improved cardiac glucose uptake, atrophy, and function, with no adverse side effects. In addition, by redirecting glucose to the heart in addition to other organs, the systemic insulin treatment lowered glucose usage by the tumor and thereby decreased tumor growth and volume. Insulin corrected the cancer-induced reduction in cardiac Akt activation and the subsequent overactivation of the proteasome and autophagy. Thus, cancer-induced systemic insulin depletion contributes to cardiac wasting and failure and may promote tumor growth. Low-dose insulin supplementation attenuates these processes and may be supportive in cardio-oncologic treatment concepts.

Targeting Amino Acid Metabolism for Molecular Imaging of Inflammation Early After Myocardial Infarction.

Acute tissue inflammation after myocardial infarction influences healing and remodeling and has been identified as a target for novel therapies. Molecular imaging holds promise for guidance of such therapies. The amino acid (11)C-methionine is a clinically approved agent which is thought to accumulate in macrophages, but not in healthy myocytes. We assessed the suitability of positron emission tomography (PET) with (11)C-methionine for imaging post-MI inflammation, from cell to mouse to man. Uptake assays demonstrated 7-fold higher (11)C-methionine uptake by polarized pro-inflammatory M1 macrophages over anti-inflammatory M2 subtypes (p<0.001). C57Bl/6 mice (n=27) underwent coronary artery ligation or no surgery. Serial (11)C-methionine PET was performed 3, 5 and 7d later. MI mice exhibited a perfusion defect in 32-50% of the left ventricle (LV). PET detected increased (11)C-methionine accumulation in the infarct territory at 3d (5.9±0.9%ID/g vs 4.7±0.9 in remote myocardium, and 2.6±0.5 in healthy mice; p<0.05 and <0.01 respectively), which declined by d7 post-MI (4.3±0.6 in infarct, 3.4±0.8 in remote; p=0.03 vs 3d, p=0.08 vs healthy). Increased (11)C-methionine uptake was associated with macrophage infiltration of damaged myocardium. Treatment with anti-integrin antibodies (anti-CD11a, -CD11b, -CD49d; 100µg) lowered macrophage content by 56% and (11)C-methionine uptake by 46% at 3d post-MI. A patient study at 3d after ST-elevation MI and early reperfusion confirmed elevated (11)C-methionine uptake in the hypoperfused myocardial region. Targeting of elevated amino acid metabolism in pro-inflammatory M1 macrophages enables PET imaging-derived demarcation of tissue inflammation after MI. (11)C-methionine-based molecular imaging may assist in the translation of novel image-guided, inflammation-targeted regenerative therapies.

[(18)F]FDG is not transported by P-glycoprotein and breast cancer resistance protein at the rodent blood-brain barrier.

INTRODUCTION: Transport of 2-[(18)F]fluoro-2-deoxy-d-glucose ([(18)F]FDG) by the multidrug efflux transporters P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) at the blood-brain barrier (BBB) may confound the interpretation of [(18)F]FDG brain PET data. Aim of this study was to assess the influence of ABCB1 and ABCG2 at the BBB on brain distribution of [(18)F]FDG in vivo by performing [(18)F]FDG PET scans in wild-type and transporter knockout mice and by evaluating changes in [(18)F]FDG brain distribution after transporter inhibition. METHODS: Dynamic small-animal PET experiments (60min) were performed with [(18)F]FDG in groups of wild-type and transporter knockout mice (Abcb1a/b((-/-)), Abcg2((-/-)) and Abcb1a/b((-/-))Abcg2((-/-))) and in wild-type rats without and with i.v. pretreatment with the known ABCB1 inhibitor tariquidar (15mg/kg, given at 2h before PET). Blood was sampled from animals from the orbital sinus vein at the end of the PET scans and measured in a gamma counter. Brain uptake of [(18)F]FDG was expressed as the brain-to-blood radioactivity concentration ratio in the last PET time frame (Kb,brain). RESULTS: Kb,brain values of [(18)F]FDG were not significantly different between different mouse types both without and with tariquidar pretreatment. The blood-to-brain transfer rate constant of [(18)F]FDG was significantly lower in tariquidar-treated as compared with vehicle-treated rats (0.350±0.025mL/min/g versus 0.416±0.024mL/min/g, p=0.026, paired t-test) but Kb,brain values were not significantly different between both rat groups. CONCLUSION: Our results show that [(18)F]FDG is not transported by Abcb1 at the mouse and rat BBB in vivo. In addition we found no evidence for Abcg2 transport of [(18)F]FDG at the mouse BBB. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Our findings imply that functional activity of ABCB1 and ABCG2 at the BBB does not need to be taken into account when interpreting brain [(18)F]FDG PET data.

Pulmonary transplantation of macrophage progenitors as effective and long-lasting therapy for hereditary pulmonary alveolar proteinosis.

Hereditary pulmonary alveolar proteinosis (herPAP) is a rare lung disease caused by mutations in the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor genes, resulting in disturbed alveolar macrophage differentiation, massive alveolar proteinosis, and life-threatening respiratory insufficiency. So far, the only effective treatment for herPAP is repetitive whole-lung lavage, a merely symptomatic and highly invasive procedure. We introduce pulmonary transplantation of macrophage progenitors as effective and long-lasting therapy for herPAP. In a murine disease model, intrapulmonary transplanted macrophage progenitors displayed selective, long-term pulmonary engraftment and differentiation into functional alveolar macrophages. A single transplantation ameliorated the herPAP phenotype for at least 9 months, resulting in significantly reduced alveolar proteinosis, normalized lung densities in chest computed tomography, and improved lung function. A significant and sustained disease resolution was also observed in a second, humanized herPAP model after intrapulmonary transplantation of human macrophage progenitors. The therapeutic effect was mediated by long-lived, lung-resident macrophages, which displayed functional and phenotypical characteristics of primary human alveolar macrophages. Our findings present the concept of organotopic transplantation of macrophage progenitors as an effective and long-lasting therapy of herPAP and may also serve as a proof of principle for other diseases, expanding current stem cell-based strategies toward potent concepts using the transplantation of differentiated cells.

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.

(R)-[(11)C]verapamil is selectively transported by murine and human P-glycoprotein at the blood-brain barrier, and not by MRP1 and BCRP.

INTRODUCTION: Positron emission tomography (PET) with [(11)C]verapamil, either in racemic form or in form of the (R)-enantiomer, has been used to measure the functional activity of the adenosine triphosphate-binding cassette (ABC) transporter P-glycoprotein (Pgp) at the blood-brain barrier (BBB). There is some evidence in literature that verapamil inhibits two other ABC transporters expressed at the BBB, i.e. multidrug resistance protein 1 (MRP1) and breast cancer resistance protein (BCRP). However, previous data were obtained with micromolar concentrations of verapamil and do not necessarily reflect the transporter selectivity of verapamil at nanomolar concentrations, which are relevant for PET experiments. The aim of this study was to assess the selectivity of verapamil, in nanomolar concentrations, for Pgp over MRP1 and BCRP. METHODS: Concentration equilibrium transport assays were performed with [(3)H]verapamil (5 nM) in cell lines expressing murine or human Pgp, human MRP1, and murine Bcrp1 or human BCRP. Paired PET scans were performed with (R)-[(11)C]verapamil in female FVB/N (wild-type), Mrp1((-/-)), Mdr1a/b((-/-)), Bcrp1((-/-)) and Mdr1a/b((-/-))Bcrp1((-/-)) mice, before and after Pgp inhibition with 15 mg/kg tariquidar. RESULTS: In vitro transport experiments exclusively showed directed transport of [(3)H]verapamil in Mdr1a- and MDR1-overexpressing cells which could be inhibited by tariquidar (0.5µM). In PET scans acquired before tariquidar administration, brain-to-blood ratio (Kb,brain) of (R)-[(11)C]verapamil was low in wild-type (1.3 ± 0.1), Mrp1((-/-)) (1.4 ± 0.1) and Bcrp1((-/-)) mice (1.8 ± 0.1) and high in Mdr1a/b((-/-)) (6.9 ± 0.8) and Mdr1a/b((-/-))Bcrp1((-/-)) mice (7.9 ± 0.5). In PET scans after tariquidar administration, Kb,brain was significantly increased in Pgp-expressing mice (wild-type: 5.0 ± 0.3-fold, Mrp1((-/-)): 3.2 ± 0.6-fold, Bcrp1((-/-)): 4.3 ± 0.1-fold) but not in Pgp knockout mice (Mdr1a/b((-/-)) and Mdr1a/b((-/-))Bcrp1((-/-))). CONCLUSION: Our combined in vitro and in vivo data demonstrate that verapamil, in nanomolar concentrations, is selectively transported by Pgp and not by MRP1 and BCRP at the BBB, which supports the use of (R)-[(11)C]verapamil or racemic [(11)C]verapamil as PET tracers of cerebral Pgp function.

Assessment of cerebral P-glycoprotein expression and function with PET by combined [11C]inhibitor and [11C]substrate scans in rats.

INTRODUCTION: The adenosine triphosphate-binding cassette (ABC) transporter P-glycoprotein (Pgp) protects the brain from accumulation of lipophilic compounds by active efflux transport across the blood-brain barrier. Changes in Pgp function/expression may occur in neurological disorders, such as epilepsy, Alzheimer's or Parkinson's disease. In this work we investigated the suitability of the radiolabeled Pgp inhibitors [(11)C]elacridar and [(11)C]tariquidar to visualize Pgp density in rat brain with PET. METHODS: Rats underwent a first PET scan with [(11)C]elacridar (n = 5) or [(11)C]tariquidar (n = 6) followed by a second scan with the Pgp substrate (R)-[(11)C]verapamil after administration of unlabeled tariquidar at a dose which half-maximally inhibits cerebral Pgp (3 mg/kg). Compartmental modeling using an arterial input function and Logan graphical analysis were used to estimate rate constants and volumes of distribution (VT) of radiotracers in different brain regions. RESULTS: Brain PET signals of [(11)C]elacridar and [(11)C]tariquidar were very low (~0.5 standardized uptake value, SUV). There was a significant negative correlation between VT and K1 (i.e. influx rate constant from plasma into brain) values of [(11)C]elacridar or [(11)C]tariquidar and VT and K1 values of (R)-[(11)C]verapamil in different brain regions which was consistent with binding of [(11)C]inhibitors to Pgp and efflux of (R)-[(11)C]verapamil by Pgp. CONCLUSION: The small Pgp binding signals obtained with [(11)C]elacridar and [(11)C]tariquidar limit the applicability of these tracers to measure cerebral Pgp density. PET tracers with higher (i.e. subnanomolar) binding affinities will be needed to visualize the low density of Pgp in brain.

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.

A novel PET protocol for visualization of breast cancer resistance protein function at the blood-brain barrier.

Breast cancer resistance protein (BCRP) is the most abundant multidrug efflux transporter at the human blood-brain barrier (BBB), restricting brain distribution of various drugs. In this study, we developed a positron emission tomography (PET) protocol to visualize Bcrp function at the murine BBB, based on the dual P-glycoprotein (P-gp)/Bcrp substrate radiotracer [(11)C]tariquidar in combination with the Bcrp inhibitor Ko143. To eliminate the contribution of P-gp efflux to [(11)C]tariquidar brain distribution, we studied mice in which P-gp was genetically knocked out (Mdr1a/b((-/-)) mice) or chemically knocked out by pretreatment with cold tariquidar. We found that [(11)C]tariquidar brain uptake increased dose dependently after administration of escalating doses of Ko143, both in Mdr1a/b((-/-)) mice and in tariquidar pretreated wild-type mice. After 15 mg/kg Ko143, the maximum increase in [(11)C]tariquidar brain uptake relative to baseline scans was 6.3-fold in Mdr1a/b((-/-)) mice with a half-maximum effect dose of 4.98 mg/kg and 3.6-fold in tariquidar (8 mg/kg) pretreated wild-type mice, suggesting that the presented protocol is sensitive to visualize a range of different functional Bcrp activities at the murine BBB. We expect that this protocol can be translated to the clinic, because tariquidar can be safely administered to humans at doses that completely inhibit cerebral P-gp.