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Abstract Background Accurate pharmacokinetic modelling in PET necessitates measurements of an input function, which ideally is acquired non-invasively from image data. For hepatic pharmacokinetic modelling two input functions need to be considered, to account for the blood supply from the hepatic artery and portal vein. Image-derived measurements at the portal vein are challenging due to its small size and image artifacts caused by respiratory motion. In this work we seek to demonstrate, using phantom experiments, how a dedicated PET/MR protocol can tackle these challenges and potentially provide input function measurements of the portal vein in a clinical setup. Methods A custom 3D printed PET/MR phantom was constructed to mimic the liver and portal vein. PET/MR acquisitions were made with emulated respiratory motion. The PET/MR imaging protocol consisted of high-resolution anatomical MR imaging of the portal vein, followed by a PET acquisition in parallel to a dedicated motion-tracking MR sequence. Motion tracking and deformation information were extracted from PET data and subsequently used in PET reconstruction to produce dynamic series of motion-free PET images. Anatomical MR images were used post PET reconstruction for partial volume correction of the input function measurements. Results Reconstruction of dynamic PET data with motion-compensation provided nearly motion-free series of PET frame data, suitable for image derived input function measurements of the portal vein. After partial volume correction, the individual input function measurements were within a 16.1% error range from the true activity in the portal vein compartment at the time of PET acquisition. Conclusion The proposed protocol demonstrates clinically feasible PET/MR imaging of the liver for pharmacokinetic studies with accurate quantification of the portal vein input function, including correction for respiratory motion and partial volume effects.

Abstract Background P-glycoprotein (P-gp) is an efflux transporter which is abundantly expressed at the blood-brain barrier (BBB) and which has been implicated in the pathophysiology of various brain diseases. The radiolabelled antiemetic drug [11C]metoclopramide is a P-gp substrate for positron emission tomography (PET) imaging of P-gp function at the BBB. To assess whether [11C]metoclopramide can detect increased P-gp function in the human brain, we employed drug-resistant temporal lobe epilepsy (TLE) as a model disease with a well characterised, regional P-gp up-regulation at the BBB. Methods Eight patients with drug-resistant (DRE) TLE, 5 seizure-free patients with drug-sensitive (DSE) focal epilepsy, and 15 healthy subjects underwent brain PET imaging with [11C]metoclopramide on a fully-integrated PET/MRI system. Concurrent with PET, arterial blood sampling was performed to generate a metabolite-corrected arterial plasma input function for kinetic modelling. The choroid plexus was outmasked on the PET images to remove signal contamination from the neighbouring hippocampus. Using a brain atlas, 10 temporal lobe sub-regions were defined and analysed with a 1-tissue-2-rate constant compartmental model to estimate the rate constants for radiotracer transfer from plasma to brain (K 1) and from brain to plasma (k 2), and the total volume of distribution (V T = K 1/k 2). Results DRE patients but not DSE patients showed significantly higher k 2 values and a trend towards lower V T values in several temporal lobe sub-regions located ipsilateral to the epileptic focus as compared to healthy subjects (k 2: hippocampus: +34%, anterior temporal lobe, medial part: +28%, superior temporal gyrus, posterior part: +21%). Conclusions [11C]Metoclopramide PET can detect a seizure-induced P-gp up-regulation in the epileptic brain. The efflux rate constant k 2 seems to be the most sensitive parameter to measure increased P-gp function with [11C]metoclopramide. Our study provides evidence that disease-induced alterations in P-gp expression at the BBB can lead to changes in the distribution of a central nervous system-active drug to the human brain, which could affect the efficacy and/or safety of drugs. [11C]Metoclopramide PET may be used to assess or predict the contribution of increased P-gp function to drug resistance and disease pathophysiology in various brain diseases. Trial registration EudraCT 2019-003137-42. Registered 28 February 2020.

Abstract Background 6-Bromo-7-[11C]methylpurine ([11C]BMP) is a radiotracer for positron emission tomography (PET) to measure multidrug resistance-associated protein 1 (MRP1) transport activity in different tissues. Previously reported radiosyntheses of [11C]BMP afforded a mixture of 7- and 9-[11C]methyl regioisomers. To prepare for clinical use, we here report an improved regioselective radiosynthesis of [11C]BMP, the results of a non-clinical toxicity study as well as human dosimetry estimates based on mouse PET data. Results [11C]BMP was synthesised by regioselective N 7 -methylation of 6-bromo-7H-purine (prepared under good manufacturing practice) with [11C]methyl triflate in presence of 2,2,6,6-tetramethylpiperidine magnesium chloride in a TRACERlab™ FX2 C synthesis module. [11C]BMP was obtained within a total synthesis time of approximately 43 min in a decay-corrected radiochemical yield of 20.5 ± 5.2%, based on starting [11C]methyl iodide, with a radiochemical purity > 99% and a molar activity at end of synthesis of 197 ± 130 GBq/μmol (n = 28). An extended single-dose toxicity study conducted in male and female Wistar rats under good laboratory practice after single intravenous (i.v.) administration of unlabelled BMP (2 mg/kg body weight) revealed no test item related adverse effects. Human dosimetry estimates, based on dynamic whole-body PET data in female C57BL/6J mice, suggested that an i.v. injected activity amount of 400 MBq of [11C]BMP will deliver an effective dose in the typical range of 11C-labelled radiotracers. Conclusions [11C]BMP can be produced in sufficient amounts and acceptable quality for clinical use. Data from the non-clinical safety evaluation showed no adverse effects and suggested that the administration of [11C]BMP will be safe and well tolerated in humans.

Abstract St. John's wort (SJW) extract, a herbal medicine with antidepressant effects, is a potent inducer of intestinal and/or hepatic cytochrome P450 (CYP) enzymes and P‐glycoprotein (P‐gp), which can cause clinically relevant drug interactions. It is currently not known whether SJW can also induce P‐gp activity at the human blood–brain barrier (BBB), which may potentially lead to decreased brain exposure and efficacy of certain central nervous system (CNS)‐targeted P‐gp substrate drugs. In this study, we used a combination of positron emission tomography (PET) imaging and cocktail phenotyping to gain a comprehensive picture on the effect of SJW on central and peripheral P‐gp and CYP activities. Before and after treatment of healthy volunteers (n = 10) with SJW extract with a high hyperforin content (3–6%) for 12–19 days (1800 mg/day), the activity of P‐gp at the BBB was assessed by means of PET imaging with the P‐gp substrate [11C]metoclopramide and the activity of peripheral P‐gp and CYPs was assessed by administering a low‐dose phenotyping cocktail (caffeine, omeprazole, dextromethorphan, and midazolam or fexofenadine). SJW significantly increased peripheral P‐gp, CYP3A, and CYP2C19 activity. Conversely, no significant changes in the peripheral metabolism, brain distribution, and P‐gp‐mediated efflux of [11C]metoclopramide across the BBB were observed following the treatment with SJW extract. Our data suggest that SJW does not lead to significant P‐gp induction at the human BBB despite its ability to induce peripheral P‐gp and CYPs. Simultaneous intake of SJW with CNS‐targeted P‐gp substrate drugs is not expected to lead to P‐gp‐mediated drug interactions at the BBB.

Abstract Research conducted over the past 2 decades has enhanced the validity and expanded the applications of microdosing and other phase 0 approaches in drug development. Phase 0 approaches can accelerate drug development timelines and reduce attrition in clinical development by increasing the quality of candidates entering clinical development and by reducing the time to “go‐no‐go” decisions. This can be done by adding clinical trial data (both healthy volunteers and patients) to preclinical candidate selection, and by applying methodological and operational advantages that phase 0 have over traditional approaches. The main feature of phase 0 approaches is the limited, subtherapeutic exposure to the test article. This means a reduced risk to research volunteers, and reduced regulatory requirements, timelines, and costs of first‐in‐human (FIH) testing. Whereas many operational aspects of phase 0 approaches are similar to those of other early phase clinical development programs, they have some unique strategic, regulatory, ethical, feasibility, economic, and cultural aspects. Here, we provide a guidance to these operational aspects and include case studies to highlight their potential impact in a range of clinical development scenarios.

Abstract Background To better understand the etiology and pathomechanisms of Alzheimer’s disease, several transgenic animal models that overexpress human tau or human amyloid-beta (Aβ) have been developed. In the present study, we generated a novel transgenic rat model by cross-breeding amyloid precursor protein (APP) rats with tau rats. We characterized this model by performing positron emission tomography scans combined with immunofluorescent labeling and cerebrospinal fluid analyses. Methods APP/Tau rats were generated by cross-breeding male McGill-R-Thy1-APP transgenic rats with female hTau-40/P301L transgenic rats. APP/Tau double transgenic rats and non-transgenic (ntg) littermates aged 7, 13, and 21 months were subjected to dynamic [11C] PiB scan and dynamic [18F]THK-5317 scans. For regional brain analysis, a template was generated from anatomical MR images of selected animals, which was co-registered with the PET images. Regional analysis was performed by application of the simplified reference tissue model ([11C]PiB data), whereas [18F]THK-5317 data were analyzed using a 2-tissue compartment model and Logan graphical analysis. In addition, immunofluorescent labeling (tau, amyloid) and cerebrospinal fluid analyses were performed. Results [11C]PiB binding potential (BP ND ) and [18F]THK-5317 volume of distribution (V T ) showed an increase with age in several brain regions in the APP/Tau group but not in the ntg control group. Immunohistochemical analysis of brain slices of PET-scanned animals revealed a positive correlation between Aβ labeling and [11C]PiB regional BP ND . Tau staining yielded a trend towards higher levels in the cortex and hippocampus of APP/Tau rats compared with ntg littermates, but without reaching statistical significance. No correlation was found between tau immunofluorescence labeling results and the respective [18F]THK-5317 V T values. Conclusions We thoroughly characterized a novel APP/Tau rat model using combined PET imaging and immunofluorescence analysis. We observed an age-related increase in [11C]PiB and [18F]THK-5317 binding in several brain regions in the APP/Tau group but not in the ntg group. Although we were able to reveal a positive correlation between amyloid labeling and [11C]PiB regional brain uptake, we observed relatively low human tau and amyloid fibril expression levels and a somewhat unstable brain pathology which questions the utility of this animal model for further studies.

[11C]metoclopramide PET imaging provides a sensitive and translational tool to explore P-glycoprotein (P-gp) function at the blood-brain barrier (BBB). Patients with neurological diseases are often treated with cytochrome (CYP) modulators which may impact the plasma and brain kinetics of [11C]metoclopramide. The impact of the CYP inducer carbamazepine or the CYP inhibitor ritonavir on the brain and plasma kinetics of [11C]metoclopramide was investigated in rats. Data obtained in a control group were compared with groups that were either orally pretreated with carbamazepine (45 mg/kg twice a day for 7 days before PET) or ritonavir (20 mg/kg, 3 h before PET) (n = 4 per condition). Kinetic modelling was performed to estimate the brain penetration (VT) of [11C]metoclopramide. CYP induction or inhibition had negligible impact on the plasma kinetics and metabolism of [11C]metoclopramide. Moreover, carbamazepine neither impacted the brain kinetics nor VT of [11C]metoclopramide (p > 0.05). However, ritonavir significantly increased VT (p < 0.001), apparently behaving as an inhibitor of P-gp at the BBB. Our data suggest that treatment with potent CYP inducers such as carbamazepine does not bias the estimation of P-gp function at the BBB with [11C]metoclopramide PET. This supports further use of [11C]metoclopramide for studies in animals and patients treated with CYP inducers.

Abstract Background P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) are two efflux transporters expressed at the blood–brain barrier which effectively restrict the brain distribution of the majority of currently known anticancer drugs. High-grade brain tumors often possess a disrupted blood–brain tumor barrier (BBTB) leading to enhanced accumulation of magnetic resonance imaging contrast agents, and possibly anticancer drugs, as compared to normal brain. In contrast to high-grade brain tumors, considerably less information is available with respect to BBTB integrity in lower grade brain tumors. Materials and methods We performed positron emission tomography imaging with the radiolabeled ABCB1 inhibitor [11C]tariquidar, a prototypical ABCB1/ABCG2 substrate, in seven patients with non-contrast -enhancing brain tumors (WHO grades I–III). In addition, ABCB1 and ABCG2 levels were determined in surgically resected tumor tissue of four patients using quantitative targeted absolute proteomics. Results Brain distribution of [11C]tariquidar was found to be very low across the whole brain and not significantly different between tumor and tumor-free brain tissue. Only one patient showed a small area of enhanced [11C]tariquidar uptake within the brain tumor. ABCG2/ABCB1 ratios in surgically resected tumor tissue (1.4 ± 0.2) were comparable to previously reported ABCG2/ABCB1 ratios in isolated human micro-vessels (1.3), which suggested that no overexpression of ABCB1 or ABCG2 occurred in the investigated tumors. Conclusions Our data suggest that the investigated brain tumors had an intact BBTB, which is impermeable to anticancer drugs, which are dual ABCB1/ABCG2 substrates. Therefore, effective drugs for antitumor treatment should have high passive permeability and lack ABCB1/ABCG2 substrate affinity. Trial registration European Union Drug Regulating Authorities Clinical Trials Database (EUDRACT), 2011-004189-13. Registered on 23 February 2012, https://www.clinicaltrialsregister.eu/ctr-search/search?query=2011-004189-13.

Abstract Background [11C]Metoclopramide is a new radiotracer for investigating the activity of P-glycoprotein at the blood-brain barrier. A highly stable and reproducible radiosynthesis is a prerequisite for clinical studies applying [11C]metoclopramide or other 11C-labelled radiotracers, therefore all potential pitfalls must be identified and monitored to allow a stable process. Results Long-term production (n = 94 in a time range of approximately 2 years) of [11C]metoclopramide synthesized on two commercially available synthesizers yielded 3.9 ± 2.0 GBq of product with a molar activity of 132 ± 164 GBq/μmol and an overall success rate of 93%. During all successful productions, the product quality was in accordance with the recommendations of the European Pharmacopoeia. The most common pitfalls that were identified for the radiosynthesis included poor turnover into [11C]CH3OTf, decomposition of the solvent or insufficient semi-preparative HPLC performance. Conclusion The study provides long-term insight in the improved, robust and stable preparation of [11C]metoclopramide for human use.

The widely expressed and poly-specific ABC transporters breast cancer resistance protein (ABCG2) and P-glycoprotein (ABCB1) are co-localized at the blood-brain barrier (BBB) and have shown to limit the brain distribution of several clinically used ABCB1/ABCG2 substrate drugs. It is currently not known to which extent these transporters, which are also expressed at the blood-retinal barrier (BRB), may limit drug distribution to the human eye and whether the ABCG2 reduced-function single-nucleotide polymorphism (SNP) Q141K (c.421C > A) has an impact on retinal drug distribution. Ten healthy male volunteers (five subjects with the c.421CC and c.421CA genotype, respectively) underwent two consecutive positron emission tomography (PET) scans after intravenous injection of the model ABCB1/ABCG2 substrate [11C]tariquidar. The second PET scan was performed with concurrent intravenous infusion of unlabelled tariquidar to inhibit ABCB1 in order to specifically reveal ABCG2 function.In response to ABCB1 inhibition with unlabelled tariquidar, ABCG2 c.421C > A genotype carriers showed significant increases (as compared to the baseline scan) in retinal radiotracer influx K1 (+62 ± 57%, p = 0.043) and volume of distribution VT (+86 ± 131%, p = 0.043), but no significant changes were observed in subjects with the c.421C > C genotype. Our results provide the first evidence that ABCB1 and ABCG2 may together limit the distribution of systemically administered ABCB1/ABCG2 substrate drugs to the human retina. Functional redundancy between ABCB1 and ABCG2 appears to be compromised in carriers of the c.421C > A SNP who may therefore be more susceptible to transporter-mediated drug-drug interactions at the BRB than non-carriers.

Multidrug resistance-associated protein-1 (MRP1/ABCC1) is highly expressed in human lung tissues. Recent studies suggest that it significantly affects the pulmonary disposition of its substrates, both after pulmonary and systemic administration. To better understand the molecular mechanisms involved, we studied the expression, subcellular localization and activity of MRP1 in freshly isolated human alveolar epithelial type 2 (AT2) and type 1-like (AT1-like) cells in primary culture, and in the NCI-H441 cell line. Moreover, the effect of cigarette smoke extract (CSE) and a series of inhaled drugs on MRP1 abundance and activity was investigated in vitro. MRP1 expression levels were measured by q-PCR and immunoblot in AT2 and AT1-like cells from different donors and in several passages of the NCI-H441 cell line. The subcellular localization of the transporter was studied by confocal laser scanning microscopy and cell surface protein biotinylation. MRP1 activity was assessed by bidirectional transport and efflux experiments using the MRP1 substrate, 5(6)-carboxyfluorescein [CF; formed intracellularly from 5(6)-carboxyfluorescein-diacetate (CFDA)] in AT1-like and NCI-H441 cell monolayers. Furthermore, the effect of CSE as well as several bronchodilators and inhaled corticosteroids on MRP1 abundance and CF efflux was investigated. MRP1 protein abundance increased upon differentiation from AT2 to AT1-like phenotype, however, ABCC1 gene levels remained unchanged. MRP1 abundance in NCI-H441 cells were comparable to those found in AT1-like cells. The transporter was detected primarily in basolateral membranes of both cell types which was consistent with net basolateral efflux of CF. Likewise, bidirectional transport studies showed net apical-to-basolateral transport of CF which was sensitive to the MRP1 inhibitor MK-571. Budesonide, beclomethasone dipropionate, salbutamol sulfate, and CSE decreased CF efflux in a concentration-dependent manner. Interestingly, CSE increased MRP1 abundance, whereas budesonide, beclomethasone dipropionate, salbutamol sulfate did not have such effect. CSE and inhaled drugs can reduce MRP1 activity in vitro, which implies the transporter being a potential drug target in the treatment of chronic obstructive pulmonary disease (COPD). Moreover, MRP1 expression level, localization and activity were comparable in human AT1-like and NCI-H441 cells. Therefore, the cell line can be a useful alternative in vitro model to study MRP1 in distal lung epithelium.

Endotoxemia-induced inflammation may impact the activity of hepatocyte transporters, which control the hepatobiliary elimination of drugs and bile acids. 99mTc-mebrofenin is a non-metabolized substrate of transporters expressed at the different poles of hepatocytes. 99mTc-mebrofenin imaging was performed in rats after the injection of lipopolysaccharide (LPS). Changes in transporter expression were assessed using quantitative polymerase chain reaction of resected liver samples. Moreover, the particular impact of pharmacokinetic drug–drug interactions in the context of endotoxemia was investigated using rifampicin (40 mg/kg), a potent inhibitor of hepatocyte transporters. LPS increased 99mTc-mebrofenin exposure in the liver (1.7 ± 0.4-fold). Kinetic modeling revealed that endotoxemia did not impact the blood-to-liver uptake of 99mTc-mebrofenin, which is mediated by organic anion-transporting polypeptide (Oatp) transporters. However, liver-to-bile and liver-to-blood efflux rates were dramatically decreased, leading to liver accumulation. The transcriptomic profile of hepatocyte transporters consistently showed a downregulation of multidrug resistance-associated proteins 2 and 3 (Mrp2 and Mrp3), which mediate the canalicular and sinusoidal efflux of 99mTc-mebrofenin in hepatocytes, respectively. Rifampicin effectively blocked both the Oatp-mediated influx and the Mrp2/3-related efflux of 99mTc-mebrofenin. The additive impact of endotoxemia and rifampicin led to a 3.0 ± 1.3-fold increase in blood exposure compared with healthy non-treated animals. 99mTc-mebrofenin imaging is useful to investigate disease-associated change in hepatocyte transporter function.

Abstract Osteosarcoma (OS) is a rare tumor of the bone occurring mainly in young adults accounting for 5% of all childhood cancers. Because of the limited therapeutic options, there has been no survival improvement for OS patients in the past 40 years. The epidermal growth factor receptor (EGFR) is highly expressed in OS; however, its clinical relevance is unclear. Here, we employed an autochthonous c‐Fos‐dependent OS mouse model (H2‐c‐fosLTR) and human OS tumor biopsies for preclinical studies aimed at identifying novel biomarkers and therapeutic benefits of anti‐EGFR therapies. We show that EGFR deletion/inhibition results in reduced tumor formation in H2‐c‐fosLTR mice by directly inhibiting the proliferation of cancer‐initiating osteoblastic cells by a mechanism involving RSK2/CREB‐dependent c‐Fos expression. Furthermore, OS patients with co‐expression of EGFR and c‐Fos exhibit reduced overall survival. Preclinical studies using human OS xenografts revealed that only tumors expressing both EGFR and c‐Fos responded to anti‐EGFR therapy demonstrating that c‐Fos can be considered as a novel biomarker predicting response to anti‐EGFR treatment in OS patients.

Abstract Background Erlotinib is an epidermal growth factor receptor (EGFR)-targeting tyrosine kinase inhibitor approved for treatment of non-small cell lung cancer. The wide inter-individual pharmacokinetic (PK) variability of erlotinib may impact treatment outcome and/or toxicity. Recent in vivo studies reported a nonlinear uptake transport of erlotinib into the liver, suggesting carrier-mediated system(s) to mediate its hepatobiliary clearance. Erlotinib has been identified in vitro as a substrate of organic anion-transporting polypeptide (OATP) transporters which expression does not restrict to hepatocytes and may impact the tissue uptake of erlotinib in vivo. Results The impact of rifampicin (40 mg/kg), a potent OATP inhibitor, on the liver uptake and exposure to tissues of 11C-erlotinib was investigated in rats (4 animals per group) using positron emission tomography (PET) imaging. Tissue pharmacokinetics (PK) and corresponding exposure (area under the curve, AUC) were assessed in the liver, kidney cortex, abdominal aorta (blood pool) and the lungs. The plasma PK of parent 11C-erlotinib was also measured using arterial blood sampling to estimate the transfer rate constant (k uptake) of 11C-erlotinib from plasma into different tissues. PET images unveiled the predominant distribution of 11C-erlotinib-associated radioactivity to the liver, which gradually moved to the intestine, thus highlighting hepatobiliary clearance. 11C-erlotinib also accumulated in the kidney cortex. Rifampicin did not impact AUCaorta but reduced k uptake, liver (p

Abstract Background [11C]erlotinib has been proposed as a PET tracer to visualize the mutational status of the epidermal growth factor receptor (EGFR) in cancer patients. For clinical use, a stable, reproducible and high-yielding radiosynthesis method is a prerequisite. In this work, two production schemes for [11C]erlotinib applied in a set of preclinical and clinical studies, starting from either [11C]CH4 or [11C]CO2, are presented and compared in terms of radiochemical yields, molar activities and overall synthesis time. In addition, a time-efficient RP-HPLC method for quality control is presented, which requires not more than 1 min. Results [11C]erlotinib was reliably produced applying both methods with decay-corrected radiochemical yields of 13.4 ± 6.2% and 16.1 ± 4.9% starting from in-target produced [11C]CO2 and [11C]CH4, respectively. Irradiation time for the production of [11C]CO2 was higher in order to afford final product amounts sufficient for patient application. Overall synthesis time was comparable, mostly attributable to adaptions in the semi-preparative HPLC protocol. Molar activities were 1.8-fold higher for the method starting from [11C]CH4 (157 ± 68 versus 88 ± 57 GBq/μmol at the end of synthesis). Conclusions This study compared two synthetic protocols for the production of [11C]erlotinib with in-target produced [11C]CO2 or [11C]CH4. Both methods reliably yielded sufficiently high product amounts for preclinical and clinical use.

P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are co-localized at the blood–brain barrier, where they display functional redundancy to restrict the brain distribution of dual P-gp/BCRP substrate drugs. We used positron emission tomography (PET) with the metabolically stable P-gp/BCRP substrates [11C]tariquidar, [11C]erlotinib, and [11C]elacridar to assess whether a similar functional redundancy as at the BBB exists in the liver, where both transporters mediate the biliary excretion of drugs. Wild-type, Abcb1a/b(−/−), Abcg2(−/−), and Abcb1a/b(−/−)Abcg2(−/−) mice underwent dynamic whole-body PET scans after i.v. injection of either [11C]tariquidar, [11C]erlotinib, or [11C]elacridar. Brain uptake of all three radiotracers was markedly higher in Abcb1a/b(−/−)Abcg2(−/−) mice than in wild-type mice, while only moderately changed in Abcb1a/b(−/−) and Abcg2(−/−) mice. The transfer of radioactivity from liver to excreted bile was significantly lower in Abcb1a/b(−/−)Abcg2(−/−) mice and almost unchanged in Abcb1a/b(−/−) and Abcg2(−/−) mice (with the exception of [11C]erlotinib, for which biliary excretion was also significantly reduced in Abcg2(−/−) mice). Our data provide evidence for redundancy between P-gp and BCRP in controlling both the brain distribution and biliary excretion of dual P-gp/BCRP substrates and highlight the utility of PET as an upcoming tool to assess the effect of transporters on drug disposition at a whole-body level.

Abstract Amyloid-β (Aβ) deposition is one of the hallmarks of the amyloid hypothesis in Alzheimer’s disease (AD). Mouse models using APP-transgene overexpression to generate amyloid plaques have shown to model only certain parts of the disease. The extent to which the data from mice can be transferred to man remains controversial. Several studies have shown convincing treatment results in reducing Aβ and enhancing cognition in mice but failed totally in human. One model-dependent factor has so far been almost completely neglected: the endogenous expression of mouse APP and its effects on the transgenic models and the readout for therapeutic approaches. Here, we report that hAPP-transgenic models of amyloidosis devoid of endogenous mouse APP expression (mAPP-knockout / mAPPko) show increased amounts and higher speed of Aβ deposition than controls with mAPP. The number of senile plaques and the level of aggregated hAβ were elevated in mAPPko mice, while the deposition in cortical blood vessels was delayed, indicating an alteration in the general aggregation propensity of hAβ together with endogenous mAβ. Furthermore, the cellular response to Aβ deposition was modulated: mAPPko mice developed a pronounced and age-dependent astrogliosis, while microglial association to amyloid plaques was diminished. The expression of human and murine aggregation-prone proteins with differing amino acid sequences within the same mouse model might not only alter the extent of deposition but also modulate the route of pathogenesis, and thus, decisively influence the study outcome, especially in translational research.

Organic anion-transporting polypeptide 2B1 (OATP2B1) is co-localized with OATP1B1 and OATP1B3 in the basolateral hepatocyte membrane, where it is thought to contribute to the hepatic uptake of drugs. We characterized a novel Slco2b1(-/-) mouse model using positron emission tomography (PET) imaging with [11C]erlotinib (a putative OATP2B1-selective substrate) and planar scintigraphic imaging with [99mTc]mebrofenin (an OATP1B1/1B3 substrate, which is not transported by OATP2B1). Dynamic 40-min scans were performed after intravenous injection of either [11C]erlotinib or [99mTc]mebrofenin in wild-type and Slco2b1(-/-) mice. A pharmacokinetic model was used to estimate the hepatic uptake clearance (CL1) and the rate constants for transfer of radioactivity from the liver to the blood (k2) and excreted bile (k3). CL1 was significantly reduced in Slco2b1(-/-) mice for both radiotracers (p < 0.05), and k2 was significantly lower (p < 0.01) in Slco2b1(-/-) mice for [11C]erlotinib, but not for [99mTc]mebrofenin. Our data support previous evidence that OATP transporters may contribute to the hepatic uptake of [11C]erlotinib. However, the decreased hepatic uptake of the OATP1B1/1B3 substrate [99mTc]mebrofenin in Slco2b1(-/-) mice questions the utility of this mouse model to assess the relative contribution of OATP2B1 to the liver uptake of drugs which are substrates of multiple OATPs.

The multidrug resistance-associated protein 2 (MRP2) mediates the biliary excretion of drugs and metabolites. [99mTc]mebrofenin may be employed as a probe for hepatic MRP2 activity because its biliary excretion is predominantly mediated by this transporter. As the liver uptake of [99mTc]mebrofenin depends on organic anion-transporting polypeptide (OATP) activity, a safe protocol for targeted inhibition of hepatic MRP2 is needed to study the intrinsic role of each transporter system. Diltiazem (DTZ) and cyclosporin A (CsA) were first confirmed to be potent MRP2 inhibitors in vitro. Dynamic acquisitions were performed in rats (n = 5–6 per group) to assess the kinetics of [99mTc]mebrofenin in the liver, intestine and heart-blood pool after increasing doses of inhibitors. Their impact on hepatic blood flow was assessed using Doppler ultrasound (n = 4). DTZ (s.c., 10 mg/kg) and low-dose CsA (i.v., 0.01 mg/kg) selectively decreased the transfer of [99mTc]mebrofenin from the liver to the bile (k3). Higher doses of DTZ and CsA did not further decrease k3 but dose-dependently decreased the uptake (k1) and backflux (k2) rate constants between blood and liver. High dose of DTZ (i.v., 3 mg/kg) but not CsA (i.v., 5 mg/kg) significantly decreased the blood flow in the portal vein and hepatic artery. Targeted pharmacological inhibition of hepatic MRP2 activity can be achieved in vivo without impacting OATP activity and liver blood flow. Clinical studies are warranted to validate [99mTc]mebrofenin in combination with low-dose CsA as a novel substrate/inhibitor pair to untangle the role of OATP and MRP2 activity in liver diseases.

The purpose of this study was to test the feasibility of migrating a quantitative brain imaging protocol from a positron emission tomography (PET)-only system to an integrated PET/MR system. Potential differences in both absolute radiotracer concentration as well as in the derived kinetic parameters as a function of PET system choice have been investigated. Five healthy volunteers underwent dynamic (R)-[11C]verapamil imaging on the same day using a GE-Advance (PET-only) and a Siemens Biograph mMR system (PET/MR). PET-emission data were reconstructed using a transmission-based attenuation correction (AC) map (PET-only), whereas a standard MR-DIXON as well as a low-dose CT AC map was applied to PET/MR emission data. Kinetic modeling based on arterial blood sampling was performed using a 1-tissue-2-rate constant compartment model, yielding kinetic parameters (K1 and k2) and distribution volume (VT). Differences for parametric values obtained in the PET-only and the PET/MR systems were analyzed using a 2-way Analysis of Variance (ANOVA). Comparison of DIXON-based AC (PET/MR) with emission data derived from the PET-only system revealed average inter-system differences of −33 ± 14% (p < 0.05) for the K1 parameter and −19 ± 9% (p < 0.05) for k2. Using a CT-based AC for PET/MR resulted in slightly lower systematic differences of −16 ± 18% for K1 and −9 ± 10% for k2. The average differences in VT were −18 ± 10% (p < 0.05) for DIXON- and −8 ± 13% for CT-based AC. Significant systematic differences were observed for kinetic parameters derived from emission data obtained from PET/MR and PET-only imaging due to different standard AC methods employed. Therefore, a transfer of imaging protocols from PET-only to PET/MR systems is not straightforward without application of proper correction methods.Clinical Trial Registration:www.clinicaltrialsregister.eu, identifier 2013-001724-19

Much has been said about the increasing number of demented patients and the main risk factor ‘age’. Frustratingly, we do not know the precise pattern and all modulating factors that provoke the pathologic changes in the brains of affected elderly. We have to diagnose early to be able to stop the progression of diseases that irreversibly destroy brain substance. Familiar AD cases have mislead some researchers for almost 20 years, which has unfortunately narrowed the scientific understanding and has, thus, lead to insufficient funding of independent approaches. Therefore, basic researchers hardly have been able to develop causative treatments and clinicians still do not have access to prognostic and early diagnostic tools.During the recent years it became clear that insufficient Aβ export, physiologically facilitated by the ABC transporter superfamily at the brain’s barriers, plays a fundamental role in disease initiation and progression. Furthermore, export mechanisms that are deficient in affected elderly are new targets for activation and, thus, treatment, but ideally also for prevention. In sporadic AD disturbed clearance of β-amyloid from the brain is so far the most important factor for its accumulation in the parenchyma and vessel walls. Here, we review findings about the contribution of ABC transporters and of the perivascular drainage/glymphatic system on β-amyloid clearance. We highlight their potential value for innovative early diagnostics using PET and describe recently described, effective ABC transporter-targeting agents as potential causative treatment for neurodegenerative proteopathies/dementias.