Autoradiography on deparaffinized tissue sections - A feasibility study with 68Ga-labeled PET-tracers.

Tissue available for retrospective research questions is often already paraffin-embedded for better preservation. However, in vitro autoradiography (AURA) is normally performed on cryopreserved tissue sections. We hypothesized a) that it would also be feasible with deparaffinized tissue sections, enabling the use of human paraffin-embedded tissue for in vitro AURA and b) that the results would be comparable to those obtained with corresponding cryosections. For that purpose, the clinically relevant oncological targets CXCR4, SSTR and PSMA were evaluated. In vitro AURA on deparaffinized tissue sections was feasible, but only with the two receptor ligands [68Ga]Ga-PentixaFor and [68Ga]Ga-DOTANOC. [68Ga]Ga-PSMA-11 did not show any binding on deparaffinized tissue sections, suggesting that native tissue is required for an interaction between this inhibitor and the enzyme.

If It Works, Don't Touch It? A Cell-Based Approach to Studying 2-[18F]FDG Metabolism.

The glucose derivative 2-[18F]fluoro-2-deoxy-D-glucose (2-[18F]FDG) is still the most used radiotracer for positron emission tomography, as it visualizes glucose utilization and energy demand. In general, 2-[18F]FDG is said to be trapped intracellularly as 2-[18F]FDG-6-phosphate, which cannot be further metabolized. However, increasingly, this dogma is being questioned because of publications showing metabolism beyond 2-[18F]FDG-6-phosphate and even postulating 2-[18F]FDG imaging to depend on the enzyme hexose-6-phosphate dehydrogenase in the endoplasmic reticulum. Therefore, we aimed to study 2-[18F]FDG metabolism in the human cancer cell lines HT1080, HT29 and Huh7 applying HPLC. We then compared 2-[18F]FDG metabolism with intracellular tracer accumulation, efflux and the cells' metabolic state and used a graphical Gaussian model to visualize metabolic patterns. The extent of 2-[18F]FDG metabolism varied considerably, dependent on the cell line, and was significantly enhanced by glucose withdrawal. However, the metabolic pattern was quite conserved. The most important radiometabolites beyond 2-[18F]FDG-6-phosphate were 2-[18F]FDMannose-6-phosphate, 2-[18F]FDG-1,6-bisphosphate and 2-[18F]FD-phosphogluconolactone. Enhanced radiometabolite formation under glucose reduction was accompanied by reduced efflux and mirrored the cells' metabolic switch as assessed via extracellular lactate levels. We conclude that there can be considerable metabolism beyond 2-[18F]FDG-6-phosphate in cancer cell lines and a comprehensive understanding of 2-[18F]FDG metabolism might help to improve cancer research and tumor diagnosis.

High-dose testosterone treatment reduces monoamine oxidase A levels in the human brain: A preliminary report.

The sex hormones testosterone and estradiol influence brain structure and function and are implicated in the pathogenesis, prevalence and disease course of major depression. Recent research employing gender-affirming hormone treatment (GHT) of gender dysphoric individuals and utilizing positron emission tomography (PET) indicates increased serotonin transporter binding upon high-dosages of testosterone treatment. Here, we investigated the effects of GHT on levels of monoamine oxidase A (MAO-A), another key target of antidepressant treatment. Participants underwent PET with the radioligand [11C]harmine to assess cerebral MAO-A distribution volumes (VT) before and four months after initiation of GHT. By the time this study was terminated for technical reasons, 18 transgender individuals undergoing GHT (11 transmen, TM and 7 transwomen, TW) and 17 cis-gender subjects had been assessed. Preliminary analysis of available data revealed statistically significant MAO-A VT reductions in TM under testosterone treatment in six of twelve a priori defined regions of interest (middle frontal cortex (-10%), anterior cingulate cortex (-9%), medial cingulate cortex (-10.5%), insula (-8%), amygdala (-9%) and hippocampus (-8.5%, all p<0.05)). MAO-A VT did not change in TW receiving estrogen treatment. Despite the limited sample size, pronounced MAO-A VT reduction could be observed, pointing towards a potential effect of testosterone. Considering MAO-A's central role in regulation of serotonergic neurotransmission, changes to MAO-A VT should be further investigated as a possible mechanism by which testosterone mediates risk for, symptomatology of, and treatment response in affective disorders.

PSMA Expression in 122 Treatment Naive Glioma Patients Related to Tumor Metabolism in 11C-Methionine PET and Survival.

Apart from its expression in benign and malignant prostate tissue, prostate specific membrane antigen (PSMA) was shown to be expressed specifically in the neovasculature of solid tumors. For gliomas only little information exists. Therefore, we aimed to correlate PSMA expression in gliomas to tumor metabolism by L-[S-methyl-11C]methionine (MET) PET and survival. Therefore, immunohistochemical staining (IHC) for isocitrate dehydrogenase 1-R132H (IDH1-R132H) mutation and PSMA expression was performed on the paraffin embedded tissue samples of 122 treatment-naive glioma patients. The IHC results were then related to the pre-therapeutic semiquantitative MET PET data and patients' survival. Vascular PSMA expression was observed in 26 of 122 samples and was rather specific for high-grade gliomas ([HGG] 81% of glioblastoma multiforme, 10% of WHO grade III and just 2% of grade II gliomas). Significantly higher amounts of gliomas without verifiable IDH1-R132H mutation showed vascular PSMA expression. Significantly shorter median survival times were seen for patients with vascular PSMA staining in all tumors as well as HGG only. Additionally, significantly higher numbers of PSMA staining vessels were found in tumors with high amino acid metabolic rates. Vascular PSMA expression in gliomas was seen as a high-grade specific feature associated with elevated amino acid metabolism and short survival.

Discovery of melanin-concentrating hormone receptor 1 in brown adipose tissue.

Although extensive research on brown adipose tissue (BAT) has stimulated optimism in the battle against obesity and diabetes, BAT physiology and organ crosstalk are not fully understood. Besides BAT, melanin-concentrating hormone (MCH) and its receptor (MCHR1) play an important role in energy homeostasis. Because of the link between hypothalamic MCH neurons and sympathetic BAT activation via ß-adrenoceptors, we investigated the expression and physiological role of the MCHR1 in BAT. MCHR1 was detected in rodent and human BAT with RT-qPCR and western blot analyses. In vivo imaging in rats used the glucose analog [18 F]FDG and the MCHR1-tracer [11 C]SNAP-7941. We found that the ß3-adrenoceptor (ADRB3) agonist CL316,243 increased [11 C]SNAP-7941 uptake in BAT. Additionally, a pharmacological concentration of SNAP-7941-a low-affinity ADRB3 ligand-stimulated [18 F]FDG uptake, reflecting BAT activation. In cultured human adipocytes, CL316,243 induced MCHR1 expression, further supporting a direct interaction between MCHR1 and ADRB3. These findings characterized MCHR1 expression in rodent and human BAT for the first time, including in vitro and in vivo data demonstrating a link between MCHR1 and the ß3-adrenergic system. The presence of MCHR1 in BAT emphasizes the role of BAT in energy homeostasis and may help uncover treatment approaches for obesity.

Conditional Generative Adversarial Networks Aided Motion Correction of Dynamic 18F-FDG PET Brain Studies.

This work set out to develop a motion-correction approach aided by conditional generative adversarial network (cGAN) methodology that allows reliable, data-driven determination of involuntary subject motion during dynamic 18F-FDG brain studies. Methods: Ten healthy volunteers (5 men/5 women; mean age ± SD, 27 ± 7 y; weight, 70 ± 10 kg) underwent a test-retest 18F-FDG PET/MRI examination of the brain (n = 20). The imaging protocol consisted of a 60-min PET list-mode acquisition contemporaneously acquired with MRI, including MR navigators and a 3-dimensional time-of-flight MR angiography sequence. Arterial blood samples were collected as a reference standard representing the arterial input function (AIF). Training of the cGAN was performed using 70% of the total datasets (n = 16, randomly chosen), which was corrected for motion using MR navigators. The resulting cGAN mappings (between individual frames and the reference frame [55-60 min after injection]) were then applied to the test dataset (remaining 30%, n = 6), producing artificially generated low-noise images from early high-noise PET frames. These low-noise images were then coregistered to the reference frame, yielding 3-dimensional motion vectors. Performance of cGAN-aided motion correction was assessed by comparing the image-derived input function (IDIF) extracted from a cGAN-aided motion-corrected dynamic sequence with the AIF based on the areas under the curves (AUCs). Moreover, clinical relevance was assessed through direct comparison of the average cerebral metabolic rates of glucose (CMRGlc) values in gray matter calculated using the AIF and the IDIF. Results: The absolute percentage difference between AUCs derived using the motion-corrected IDIF and the AIF was (1.2% + 0.9%). The gray matter CMRGlc values determined using these 2 input functions differed by less than 5% (2.4% + 1.7%). Conclusion: A fully automated data-driven motion-compensation approach was established and tested for 18F-FDG PET brain imaging. cGAN-aided motion correction enables the translation of noninvasive clinical absolute quantification from PET/MR to PET/CT by allowing the accurate determination of motion vectors from the PET data itself.

Fully Integrated PET/MR Imaging for the Assessment of the Relationship Between Functional Connectivity and Glucose Metabolic Rate.

In the past, determination of absolute values of cerebral metabolic rate of glucose (CMRGlc) in clinical routine was rarely carried out due to the invasive nature of arterial sampling. With the advent of combined PET/MR imaging technology, CMRGlc values can be obtained non-invasively, thereby providing the opportunity to take advantage of fully quantitative data in clinical routine. However, CMRGlc values display high physiological variability, presumably due to fluctuations in the intrinsic activity of the brain at rest. To reduce CMRGlc variability associated with these fluctuations, the objective of this study was to determine whether functional connectivity measures derived from resting-state fMRI (rs-fMRI) could be used to correct for these fluctuations in intrinsic brain activity. METHODS: We studied 10 healthy volunteers who underwent a test-retest dynamic [18F]FDG-PET study using a fully integrated PET/MR system (Siemens Biograph mMR). To validate the non-invasive derivation of an image-derived input function based on combined analysis of PET and MR data, arterial blood samples were obtained. Using the arterial input function (AIF), parametric images representing CMRGlc were determined using the Patlak graphical approach. Both directed functional connectivity (dFC) and undirected functional connectivity (uFC) were determined between nodes in six major networks (Default mode network, Salience, L/R Executive, Attention, and Sensory-motor network) using either a bivariate-correlation (R coefficient) or a Multi-Variate AutoRegressive (MVAR) model. In addition, the performance of a regional connectivity measure, the fractional amplitude of low frequency fluctuations (fALFF), was also investigated. RESULTS: The average intrasubject variability for CMRGlc values between test and retest was determined as (14 ±8%) with an average inter-subject variability of 25% at test and 15% at retest. The average CMRGlc value (umol/100 g/min) across all networks was 39 ±10 at test and increased slightly to 43 ±6 at retest. The R, MVAR and fALFF coefficients showed relatively large test-retest variability in comparison to the inter-subjects variability, resulting in poor reliability (intraclass correlation in the range of 0.11-0.65). More importantly, no significant relationship was found between the R coefficients (for uFC), MVAR coefficients (for dFC) or fALFF and corresponding CMRGlc values for any of the six major networks. DISCUSSION: Measurement of functional connectivity within established brain networks did not provide a means to decrease the inter- or intrasubject variability of CMRGlc values. As such, our results indicate that connectivity measured derived from rs-fMRI acquired contemporaneously with PET imaging are not suited for correction of CMRGlc variability associated with intrinsic fluctuations of resting-state brain activity. Thus, given the observed substantial inter- and intrasubject variability of CMRGlc values, the relevance of absolute quantification for clinical routine is presently uncertain.

Utility of Absolute Quantification in Non-lesional Extratemporal Lobe Epilepsy Using FDG PET/MR Imaging.

The purpose of this study was to establish a non-invasive clinical PET/MR protocol using [18F]-labeled deoxyglucose (FDG) that provides physicians with regional metabolic rate of glucose (MRGlc) values and to clarify the contribution of absolute quantification to clinical management of patients with non-lesional extratemporal lobe epilepsy (ETLE). The study included a group of 15 patients with non-lesional ETLE who underwent a dynamic FDG PET study using a fully-integrated PET/MRI system (Siemens Biograph). FDG tracer uptake images were converted to MRGlc (µmol/100 g/min) maps using an image derived input function that was extracted based on the combined analysis of PET and MRI data. In addition, the same protocol was applied to a group of healthy controls, yielding a normative database. Abnormality maps for ETLE patients were created with respect to the normative database, defining significant hypo- or hyper-metabolic regions that exceeded ±2 SD of normal regional mean MRGlc values. Abnormality maps derived from MRGlc images of ETLE patients contributed to the localization of hypo-metabolic areas against visual readings in 53% and increased the confidence in the original clinical readings in 33% of all cases. Moreover, quantification allowed identification of hyper-metabolic areas that are associated with frequently spiking cortex, rarely acknowledged in clinical readings. Overall, besides providing some confirmatory information to visual readings, quantitative PET imaging demonstrated only a moderate impact on clinical management of patients with complex pathology that leads to epileptic seizures, failing to provide new decisive information that would have changed classification of patients from being rejected to being considered for surgical intervention.

Inhibition of Lipid Accumulation in Skeletal Muscle and Liver Cells: A Protective Mechanism of Bilirubin Against Diabetes Mellitus Type 2.

Ectopic lipid accumulation in skeletal muscle and liver drives the pathogenesis of diabetes mellitus type 2 (DMT2). Mild hyperbilirubinaemia has been repeatedly suggested to play a role in the prevention of DMT2 and is known for its capacity to shape an improved lipid phenotype in humans and in animals. To date, the effect of bilirubin on lipid accumulation in tissues that are prone to ectopic lipid deposition is unclear. Therefore, we analyzed the effect of bilirubin on lipid accumulation in skeletal muscle and liver cell lines. C2C12 skeletal mouse muscle and HepG2 human liver cells were treated with physiological concentrations of free fatty acids (FFA) (0.5 mM and 1 mM) and unconjugated bilirubin (UCB) (17.1 and 55 µM). The intracellular presence of UCB upon exogenous UCB administration was confirmed by HPLC and the lipid accumulation was assessed by using Nile red. Exposure of both cell lines to UCB significantly reduced lipid accumulation by up to 23% (p ≤ 0.001) in HepG2 and by up to 17% (p ≤ 0.01) in C2C12 cells at 0.5 and 5 h under hypoglycaemic conditions. Simultaneously, UCB slightly increased FFA uptake in HepG2 cells after 0.5 and 5 h and in C2C12 cells after 12 h as confirmed by gas chromatographic analyses of the remaining FFA content in the incubation media. The effects of UCB on lipid accumulation and uptake were abolished in the presence of higher glucose concentrations. Monitoring the uptake of a radiolabeled glucose analogue [18F]FDG: (2-deoxy-2-[18F]fluoro-D-glucose) into both cell types further indicated higher glucose consumption in the presence of UCB. In conclusion, our findings show that UCB considerably decreases lipid accumulation in skeletal muscle and liver cells within a short incubation time of max. 5 h which suggests that mildly elevated bilirubin levels could lower ectopic lipid deposition, a major key element in the pathogenesis of DMT2.

Promise of Fully Integrated PET/MRI: Noninvasive Clinical Quantification of Cerebral Glucose Metabolism.

We describe a fully automated processing pipeline to support the noninvasive absolute quantification of the cerebral metabolic rate for glucose (CMRGlc) in a clinical setting. This pipeline takes advantage of "anatometabolic" information associated with fully integrated PET/MRI. Methods: Ten healthy volunteers (5 men and /5 women; 27 ± 7 y old; 70 ± 10 kg) underwent a test-retest 18F-FDG PET/MRI examination of the brain. The imaging protocol consisted of a 60-min PET list-mode acquisition with parallel MRI acquisitions, including 3-dimensional time-of-flight MR angiography, MRI navigators, and a T1-weighted MRI scan. State-of-the-art MRI-based attenuation correction was derived from T1-weighted MRI (pseudo-CT [pCT]). For validation purposes, a low-dose CT scan was also performed. Arterial blood samples were collected as the reference standard (arterial input function [AIF]). The developed pipeline allows the derivation of an image-derived input function (IDIF), which is subsequently used to create CMRGlc maps by means of a Patlak analysis. The pipeline also includes motion correction using the MRI navigator sequence as well as a novel partial-volume correction that accounts for background heterogeneity. Finally, CMRGlc maps are used to generate a normative database to facilitate the detection of metabolic abnormalities in future patient scans. To assess the performance of the developed pipeline, IDIFs extracted by both CT-based attenuation correction (CT-IDIF) and MRI-based attenuation correction (pCT-IDIF) were compared with the reference standard (AIF) using the absolute percentage difference between the areas under the curves as well as the absolute percentage difference in regional CMRGlc values. Results: The absolute percentage differences between the areas under the curves for CT-IDIF and pCT-IDIF were determined to be 1.4% ± 1.0% and 3.4% ± 2.6%, respectively. The absolute percentage difference in regional CMRGlc values based on CT-IDIF and pCT-IDIF differed by less than 6% from the reference values obtained from the AIF. Conclusion: By taking advantage of the capabilities of fully integrated PET/MRI, we developed a fully automated computational pipeline that allows the noninvasive determination of regional CMRGlc values in a clinical setting. This methodology might facilitate the proliferation of fully quantitative imaging into the clinical arena and, as a result, might contribute to improved diagnostic efficacy.

In vitro Radiopharmaceutical Evidence for MCHR1 Binding Sites in Murine Brown Adipocytes.

[11C]SNAP-7941 and its radiofluorinated, fluoro-ethyl derivative [18F]FE@SNAP have been developed as the first positron emission tomography tracers for melanin-concentrating hormone receptor 1 (MCHR1) imaging. Accumulation of these MCHR1 PET-tracers in rat brown adipose tissue (BAT) in vivo provided first indication of MCHR1 expression in rodent BAT. To rule out off-target binding, affinity of both MCHR1 ligands toward adrenergic beta-3 receptors (ADRB3) was examined. Further, specific binding of [11C]SNAP-7941 to brown adipocytes and effects of MCHR1 ligands on brown adipocyte activation were investigated. SNAP-7941 and FE@SNAP evinced to be highly selective toward MCHR1. [11C]SNAP-7941 binding to brown adipocytes was shown to be mainly MCHR1-specific. This data strongly indicates MCHR1 expression in rodent BAT and moreover, a peripheral, anti-obesity effect of MCHR1 antagonists directly exerted in BAT is proposed. Moreover, MCHR1 expression in murine brown adipocytes was confirmed by protein and mRNA analysis. We conclude that MCHR1 PET imaging contributes to basic research in endocrinology by elucidating the involvement of the MCH system in peripheral tissues, such as BAT.

Modeling the acute pharmacological response to selective serotonin reuptake inhibitors in human brain using simultaneous PET/MR imaging.

Pharmacological imaging of the effects of selective serotonin reuptake inhibitors (SSRI) may aid the clarification of their mechanism of action and influence treatment of highly prevalent neuropsychiatric conditions if the detected effects could be related to patient outcomes. In a randomized double-blind design, 38 healthy participants received a constant infusion of 8 mg citalopram or saline during either their first or second of two PET/MR scans. Resting-state functional MRI (fMRI) was acquired simultaneously with PET data on the binding of serotonin transporters (5-HTT) using [11C]DASB. Three different approaches for modeling of pharmacological fMRI response were tested separately. These relied on the use of regressors corresponding to (1) the drug infusion paradigm, (2) time courses of citalopram plasma concentrations and (3) changes in 5-HTT binding measured in each individual, respectively. Furthermore, the replication of results of a widely used model-free analysis method was attempted which assesses the deviation of signal in discrete time bins of fMRI data acquired after start of drug infusion. Following drug challenge, average 5-HTT occupancy was 69±7% and peak citalopram plasma levels were 111.8 ±â€¯21.1 ng/ml. None of the applied methods could detect significant differences in the pharmacological response between SSRI and placebo scans. The failed replication of SSRI effects reported in the literature despite a threefold larger sample size highlights the importance of appropriate correction for family-wise error in order to avoid spurious results in pharmacological imaging. This calls for the development of analysis methods which take regional specialization and the dynamics of brain activity into account.

Impact of P-Glycoprotein Function on the Brain Kinetics of the Weak Substrate 11C-Metoclopramide Assessed with PET Imaging in Humans.

PET with avid substrates of P-glycoprotein (ABCB1) provided evidence of the role of this efflux transporter in effectively restricting the brain penetration of its substrates across the human blood-brain barrier (BBB). This may not reflect the situation for weak ABCB1 substrates including several antidepressants, antiepileptic drugs, and neuroleptics, which exert central nervous system effects despite being transported by ABCB1. We performed PET with the weak ABCB1 substrate 11C-metoclopramide in humans to elucidate the impact of ABCB1 function on its brain kinetics. Methods: Ten healthy male subjects underwent 2 consecutive 11C-metoclopramide PET scans without and with ABCB1 inhibition using cyclosporine A (CsA). Pharmacokinetic modeling was performed to estimate the total volume of distribution (VT) and the influx (K1) and efflux (k2) rate constants between plasma and selected brain regions. Furthermore, 11C-metoclopramide washout from the brain was estimated by determining the elimination slope (kE,brain) of the brain time-activity curves. Results: In baseline scans, 11C-metoclopramide showed appreciable brain distribution (VT = 2.11 ± 0.33 mL/cm3). During CsA infusion, whole-brain gray matter VT and K1 were increased by 29% ± 17% and 9% ± 12%, respectively. K2 was decreased by 15% ± 5%, consistent with a decrease in kE,brain (-32% ± 18%). The impact of CsA on outcome parameters was significant and similar across brain regions except for the pituitary gland, which is not protected by the BBB. Conclusion: Our results show for the first time that ABCB1 does not solely account for the "barrier" property of the BBB but also acts as a detoxifying system to limit the overall brain exposure to its substrates at the human blood-brain interface.

Towards quantitative [18F]FDG-PET/MRI of the brain: Automated MR-driven calculation of an image-derived input function for the non-invasive determination of cerebral glucose metabolic rates.

Absolute quantification of PET brain imaging requires the measurement of an arterial input function (AIF), typically obtained invasively via an arterial cannulation. We present an approach to automatically calculate an image-derived input function (IDIF) and cerebral metabolic rates of glucose (CMRGlc) from the [18F]FDG PET data using an integrated PET/MRI system. Ten healthy controls underwent test-retest dynamic [18F]FDG-PET/MRI examinations. The imaging protocol consisted of a 60-min PET list-mode acquisition together with a time-of-flight MR angiography scan for segmenting the carotid arteries and intermittent MR navigators to monitor subject movement. AIFs were collected as the reference standard. Attenuation correction was performed using a separate low-dose CT scan. Assessment of the percentage difference between area-under-the-curve of IDIF and AIF yielded values within ±5%. Similar test-retest variability was seen between AIFs (9 ± 8) % and the IDIFs (9 ± 7) %. Absolute percentage difference between CMRGlc values obtained from AIF and IDIF across all examinations and selected brain regions was 3.2% (interquartile range: (2.4-4.3) %, maximum < 10%). High test-retest intravariability was observed between CMRGlc values obtained from AIF (14%) and IDIF (17%). The proposed approach provides an IDIF, which can be effectively used in lieu of AIF.

A Proof-of-Concept Study to Inhibit ABCG2- and ABCB1-Mediated Efflux Transport at the Human Blood-Brain Barrier.

The adenosine triphosphate-binding cassette transporters P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) are 2 efflux transporters at the blood-brain barrier (BBB) that effectively restrict brain distribution of dual ABCB1/ABCG2 substrate drugs, such as tyrosine kinase inhibitors. Pharmacologic inhibition of ABCB1/ABCG2 may improve the efficacy of dual-substrate drugs for treatment of brain tumors, but no marketed ABCB1/ABCG2 inhibitors are currently available. In the present study, we examined the potential of supratherapeutic-dose oral erlotinib to inhibit ABCB1/ABCG2 activity at the human BBB. Methods: Healthy men underwent 2 consecutive PET scans with 11C-erlotinib: a baseline scan and a second scan either with concurrent intravenous infusion of the ABCB1 inhibitor tariquidar (3.75 mg/min, n = 5) or after oral intake of single ascending doses of erlotinib (300 mg, n = 7; 650 mg, n = 8; or 1,000 mg, n = 2). Results: Although tariquidar administration had no effect on 11C-erlotinib brain distribution, oral erlotinib led, at the 650-mg dose, to significant increases in volume of distribution (23% ± 13%, P = 0.008), influx rate constant of radioactivity from plasma into brain (58% ± 26%, P = 0.008), and area under the brain time-activity curve (78% ± 17%, P = 0.008), presumably because of combined partial saturation of ABCG2 and ABCB1 activity. Inclusion of further subjects into the 1,000-mg dose group was precluded by adverse skin events (rash). Conclusion: Supratherapeutic-dose erlotinib may be used to enhance brain delivery of ABCB1/ABCG2 substrate anticancer drugs, but its clinical applicability for continuous ABCB1/ABCG2 inhibition at the BBB may be limited by safety concerns.

Effect of Rifampicin on the Distribution of [11C]Erlotinib to the Liver, a Translational PET Study in Humans and in Mice.

Organic anion-transporting polypeptides (OATPs) mediate the uptake of various drugs from blood into the liver in the basolateral membrane of hepatocytes. Positron emission tomography (PET) is a potentially powerful tool to assess the activity of hepatic OATPs in vivo, but its utility critically depends on the availability of transporter-selective probe substrates. We have shown before that among the three OATPs expressed in hepatocytes (OATP1B1, OATP1B3, and OATP2B1), [11C]erlotinib is selectively transported by OATP2B1. In contrast to OATP1B1 and OATP1B3, OATP2B1 has not been thoroughly explored yet, and no specific probe substrates are currently available. To assess if the prototypical OATP inhibitor rifampicin can inhibit liver uptake of [11C]erlotinib in vivo, we performed [11C]erlotinib PET scans in six healthy volunteers without and with intravenous pretreatment with rifampicin (600 mg). In addition, FVB mice underwent [11C]erlotinib PET scans without and with concurrent intravenous infusion of high-dose rifampicin (100 mg/kg). Rifampicin caused a moderate reduction in the liver distribution of [11C]erlotinib in humans, while a more pronounced effect of rifampicin was observed in mice, in which rifampicin plasma concentrations were higher than in humans. In vitro uptake experiments in an OATP2B1-overexpressing cell line indicated that rifampicin inhibited OATP2B1 transport of [11C]erlotinib in a concentration-dependent manner with a half-maximum inhibitory concentration of 72.0 ± 1.4 µM. Our results suggest that rifampicin-inhibitable uptake transporter(s) contributed to the liver distribution of [11C]erlotinib in humans and mice and that [11C]erlotinib PET in combination with rifampicin may be used to measure the activity of this/these uptake transporter(s) in vivo. Furthermore, our data suggest that a standard clinical dose of rifampicin may exert in vivo a moderate inhibitory effect on hepatic OATP2B1.

An Overview of PET Radiochemistry, Part 1: The Covalent Labels 18F, 11C, and 13N.

This continuing educational article introduces the radiochemistry of PET tracers that exhibit a covalently bound radiolabel with the nuclides 11C, 13N, and 18F. The overall process of PET tracer production is explained, starting from the production of the radionuclide in a cyclotron; followed by the automatization process of the radiosynthesis, including the necessary steps for the respective synthesis; and finalized with the requirements for quality control.

A new method measuring the interaction of radiotracers with the human P-glycoprotein (P-gp) transporter.

In drug development, biomarkers for cerebral applications have a lower success rate compared to cardiovascular drugs or tumor therapeutics. One reason is the missing blood brain barrier penetration, caused by the tracer's interaction with efflux transporters such as the P-gp (MDR1 or ABCB1). Aim of this study was the development of a reliable model to measure the interaction of radiotracers with the human efflux transporter P-gp in parallel to the radiolabeling process. LigandTracer® Technology was used with the wildtype cell line MDCKII and the equivalent cell line overexpressing human P-gp (MDCKII-hMDR1). The method was evaluated based on established PET tracers with known interaction with the human P-gp transporter and in nanomolar concentration (15 nM). [11C]SNAP-7941 and [18F]FE@SNAP were used as P-gp substrates by comparing the real-time model with an uptake assay and µPET images. [11C]DASB [11C]Harmine, [18F]FMeNER,[18F]FE@SUPPY and [11C]Me@HAPTHI were used as tracers without interactions with P-gp in vitro. However, [11C]Me@HAPTHI shows a significant increase in SUV levels after blocking with Tariquidar. The developed real-time kinetic model uses directly PET tracers in a compound concentration, which is reflecting the in vivo situation. This method may be used at an early stage of radiopharmaceutical development to measure interactions to P-gp before conducting animal experiments.

Expanding LogP: Present possibilities.

INTRODUCTION: Due to the high candidate exclusion rate during a drug development process, an early prediction of the pharmacokinetic behavior would be needed. Accordingly, high performance bioaffinity chromatography (HPBAC) approaches are growing in popularity, however, there is a lack of knowledge and no consensus about the relation between HPBAC measurements, in vivo distribution and blood brain barrier (BBB) penetration behavior. With respect to radiotracers, there is almost no reference data available for plasma protein binding (PPB), permeability (Pm) and the membrane coefficient (KIAM). Thus, this study was aimed at exploring the relevance of measuring PPB, Pm and KIAM for the prediction of BBB penetration. METHODS: Measurements of %PPB, Pm and KIAM were performed using HPBAC. In total, 113 compounds were tested, 43 with brain uptake, 30 not showing brain uptake and 40 with known interactions with efflux transporters. Additionally, ClogP and HPLC logPowpH7.4 data were collected. RESULTS: %PPB, KIAM, Pm and ClogP values were in the same range for each of the three groups. A significant difference was observed for the HPLC logPowpH7.4 between CNS penetrating drug group (CNSpos) and the non-penetrating drug group (CNSneg), as well as for the CNSneg towards the drug group interacting with efflux transporters (DRUGefflux). However, as the other experimental data, also the HPLC logPowpH7.4 showed a broad overlapping of the single values between the groupings. CONCLUSION: Experimental reference values (logP, Pm, KIAM & PPB) of commonly used PET tracers and drugs showing different BBB penetration behavior are provided. The influence of the logP on brain uptake depends strongly on the selected method. However, using a single parameter (experimental or calculated) to predict BBB penetration or for the classification of drug groups is inexpedient.

Influence of OATPs on Hepatic Disposition of Erlotinib Measured With Positron Emission Tomography.

To assess the hepatic disposition of erlotinib, we performed positron emission tomography (PET) scans with [11 C]erlotinib in healthy volunteers without and with oral pretreatment with a therapeutic erlotinib dose (300 mg). Erlotinib pretreatment significantly decreased the liver exposure to [11 C]erlotinib with a concomitant increase in blood exposure, pointing to the involvement of a carrier-mediated hepatic uptake mechanism. Using cell lines overexpressing human organic anion-transporting polypeptides (OATPs) 1B1, 1B3, or 2B1, we show that [11 C]erlotinib is selectively transported by OATP2B1. Our data suggest that at PET microdoses hepatic uptake of [11 C]erlotinib is mediated by OATP2B1, whereas at therapeutic doses OATP2B1 transport is saturated and hepatic uptake occurs mainly by passive diffusion. We propose that [11 C]erlotinib may be used as a hepatic OATP2B1 probe substrate and erlotinib as an OATP2B1 inhibitor in clinical drug-drug interaction studies, allowing the contribution of OATP2B1 to the hepatic uptake of drugs to be revealed.