Jurgen W. A. Sijbesma

Synthesis and Preclinical Evaluation of Novel PET Probes for P-Glycoprotein Function and Expression

UNLABELLED P-glycoprotein (P-gp) is an ATP-dependent efflux pump protecting the body against xenobiotics. A P-gp substrate (7) and an inhibitor (6) were labeled with (11)C, resulting in potential tracers of P-gp function and expression. METHODS 6 and 7 were labeled using (11)CH(3)I. (11)C-verapamil was prepared as published previously, using (11)C-methyl triflate. MicroPET scans (with arterial sampling) and biodistribution studies were performed in rats pretreated with saline, cyclosporin A (CsA, 50 mg/kg), or cold 6 (15 mg/kg). RESULTS The radiochemical yields of (11)C-6 and (11)C-7 were approximately 30% with a total synthesis time of 45 min. Cerebral distribution volumes (DV) of (11)C-6 (2.35 +/- 0.11) and (11)C-7 (1.86 +/- 0.15) in saline-treated rats were higher than of (11)C-verapamil (0.64 +/- 0.12). DVs of (11)C-7 and (11)C-verapamil were significantly increased by CsA (to 5.26 +/- 0.14 and 5.85 +/- 0.32, respectively). The DV of (11)C-6 was reduced by cold 6 (to 1.65 +/- 0.03). Its uptake was also reduced (up to 67%) in several peripheral organs that express P-gp. CONCLUSIONS (11)C-7 is a novel tracer of P-gp function with higher baseline uptake than (11)C-verapamil. Upregulation of P-gp function in response to treatment (which is hard to detect with (11)C-verapamil) may be detectable using (11)C-7 and PET. Because (11)C-6 shows specific binding in target organs, this compound is the first PET tracer allowing measurement of P-gp expression.

Cardiac LXRα protects against pathological cardiac hypertrophy and dysfunction by enhancing glucose uptake and utilization

Pathological cardiac hypertrophy is characterized by a shift in metabolic substrate utilization from fatty acids to glucose, but the molecular events underlying the metabolic remodeling remain poorly understood. Here, we investigated the role of liver X receptors (LXRs), which are key regulators of glucose and lipid metabolism, in cardiac hypertrophic pathogenesis. Using a transgenic approach in mice, we show that overexpression of LXRα acts to protect the heart against hypertrophy, fibrosis, and dysfunction. Gene expression profiling studies revealed that genes regulating metabolic pathways were differentially expressed in hearts with elevated LXRα. Functionally, LXRα overexpression in isolated cardiomyocytes and murine hearts markedly enhanced the capacity for myocardial glucose uptake following hypertrophic stress. Conversely, this adaptive response was diminished in LXRα‐deficient mice. Transcriptional changes induced by LXRα overexpression promoted energy‐independent utilization of glucose via the hexosamine biosynthesis pathway, resulting in O‐GlcNAc modification of GATA4 and Mef2c and the induction of cytoprotective natriuretic peptide expression. Our results identify LXRα as a key cardiac transcriptional regulator that helps orchestrate an adaptive metabolic response to chronic cardiac stress, and suggest that modulating LXRα may provide a unique opportunity for intervening in myocyte metabolism.

PET imaging of demyelination and remyelination in the cuprizone mouse model for multiple sclerosis: a comparison between [11C]CIC and [11C]MeDAS.

Multiple Sclerosis (MS) is a neurodegenerative disease characterized by demyelinated lesions. PET imaging using specific myelin radioligands might solve the lack of a specific imaging tool for diagnosing and monitoring demyelination and remyelination in MS patients. In recent years, a few tracers have been developed for in vivo PET imaging of myelin, but they have not been fully evaluated yet. In this study, we compared [(11)C]CIC and [(11)C]MeDAS as PET tracers for monitoring demyelination and remyelination in cuprizone-fed mice. The ex vivo biodistribution of [(11)C]CIC showed decreased tracer uptake in mice fed with 0.2% cuprizone diet for 5 weeks, as compared to control mice. However, tracer uptake did not increase again after normal diet was restored for 5 weeks (remyelination). Surprisingly, in vivo PET imaging with [(11)C]CIC in cuprizone-fed mice revealed a significant reduction in whole brain tracer uptake after 5 weeks of remyelination. No correlation between ex vivo biodistribution and in vivo imaging data was found for [(11)C]CIC (r(2)=0.15, p=0.11). However, a strong correlation was found for [(11)C]MeDAS (r(2)=0.88, p<0.0001). [(11)C]MeDAS ex vivo biodistribution revealed significant decreased brain uptake in the demyelination group, as compared to controls and increased the tracer uptake after 5 weeks of remyelination. [(11)C]MeDAS images showed a low background signal and clear uptake in the brain white matter and spinal cord. Taken together, the results of this comparative study between [(11)C]CIC and [(11)C]MeDAS clearly show that [(11)C]MeDAS is the preferred PET tracer to monitor myelin changes in the brain and spinal cord in vivo.

Small-Animal PET Study of Adenosine A 1 Receptors in Rat Brain: Blocking Receptors and Raising Extracellular Adenosine

Activation of adenosine A1 receptors (A1R) in the brain causes sedation, reduces anxiety, inhibits seizures, and promotes neuroprotection. Cerebral A1R can be visualized using 8-dicyclopropylmethyl-1-11C-methyl-3-propyl-xanthine (11C-MPDX) and PET. This study aims to test whether 11C-MPDX can be used for quantitative studies of cerebral A1R in rodents. Methods: 11C-MPDX was injected (intravenously) into isoflurane-anesthetized male Wistar rats (300 g). A dynamic scan of the central nervous system was obtained, using a small-animal PET camera. A cannula in a femoral artery was used for blood sampling. Three groups of animals were studied: group 1, controls (saline-treated); group 2, animals pretreated with the A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 1 mg, intraperitoneally); and group 3, animals pretreated (intraperitoneally) with a 20% solution of ethanol in saline (2 mL) plus the adenosine kinase inhibitor 4-amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-3-yl)pyrido[2,3-d] pyrimidine dihydrochloride (ABT-702) (1 mg). DPCPX is known to occupy cerebral A1R, whereas ethanol and ABT-702 increase extracellular adenosine. Results: In groups 1 and 3, the brain was clearly visualized. High uptake of 11C-MPDX was noted in striatum, hippocampus, and cerebellum. In group 2, tracer uptake was strongly suppressed and regional differences were abolished. The treatment of group 3 resulted in an unexpected 40%–45% increase of the cerebral uptake of radioactivity as indicated by increases of PET standardized uptake value, distribution volume from Logan plot, nondisplaceable binding potential from 2-tissue-compartment model fit, and standardized uptake value from a biodistribution study performed after the PET scan. The partition coefficient of the tracer (K1/k2 from the model fit) was not altered under the study conditions. Conclusion: 11C-MPDX shows a regional distribution in rat brain consistent with binding to A1R. Tracer binding is blocked by the selective A1R antagonist DPCPX. Pretreatment of animals with ethanol and adenosine kinase inhibitor increases 11C-MPDX uptake. This increase may reflect an increased availability of A1R after acute exposure to ethanol.

PET imaging of glucose metabolism, neuroinflammation and demyelination in the lysolecithin rat model for multiple sclerosis

Background: Injection of lysolecithin in the central nervous system results in demyelination accompanied by local activation of microglia and recruitment of monocytes. Positron-emission tomography (PET) imaging, using specific tracers, may be an adequate technique to monitor these events in vivo and therefore may become a tool for monitoring disease progression in multiple sclerosis (MS) patients. Objectives: The objective of this paper is to evaluate the potential of PET imaging in monitoring local lesions, using [11C]MeDAS, [11C]PK11195 and [18F]FDG as PET tracers for myelin density, microglia activation and glucose metabolism, respectively. Methods: Sprague-Dawley rats were stereotactically injected with either 1% lysolecithin or saline in the corpus callosum and striatum of the right brain hemisphere. PET imaging was performed three days, one week and four weeks after injection. Animals were terminated after PET imaging and the brains were explanted for (immuno)histochemical analysis. Results: PET imaging was able to detect local demyelination induced by lysolecithin in the corpus callosum and striatum with [11C]MeDAS and concomitant microglia activation and monocyte recruitment with [11C]PK11195. [18F]FDG imaging demonstrated that glucose metabolism was maintained in the demyelinated lesions. Conclusion: PET imaging with multiple tracers allows simultaneous in vivo monitoring of myelin density, neuroinflammation and brain metabolism in small MS-like lesions, indicating its potential to monitor disease progression in MS patients.

Pharmacokinetic analysis of 11C-PBR28 in the rat model of herpes encephalitis (HSE): comparison with (R)-11C-PK11195

11C-PBR28 is a second-generation translocator protein (TSPO) tracer with characteristics supposedly superior to the most commonly used tracer for neuroinflammation, (R)-11C-PK11195. Despite its use in clinical research, no studies on the imaging properties and pharmacokinetic analysis of 11C-PBR28 in rodent models of neuroinflammation have been published yet. Therefore, this study aimed to evaluate 11C-PBR28 as a tool for detection and quantification of neuroinflammation in preclinical research and to compare its imaging properties with (R)-11C-PK11195. The herpes simplex encephalitis (HSE) model was used for induction of neuroinflammation in male Wistar rats. Six or 7 d after virus inoculation, a dynamic 11C-PBR28 or (R)-11C-PK11195 PET scan with arterial blood sampling was obtained. Pharmacokinetic modeling was performed on the PET data and analyzed using volumes of interest and a voxel-based approach. Volume-of-interest– and voxel-based analysis of 11C-PBR28 images showed overexpression of TSPO in brain regions known to be affected in the HSE rat model. 11C-PBR28 was metabolized faster than (R)-11C-PK11195, with a metabolic half-life in plasma of 5 and 21 min, respectively. Overall, 11C-PBR28 was more sensitive than (R)-11C-PK11195 in detecting neuroinflammation. The binding potential (BPND) of 11C-PBR28 was significantly higher (P < 0.05) in the medulla (176%), pons (146%), midbrain (101%), hippocampus (85%), thalamus (73%), cerebellum (54%), and hypothalamus (49%) in HSE rats than in control rats, whereas (R)-11C-PK11195 showed a higher BPND only in the medulla (32%). The BPND in control animals was not significantly different between tracers, suggesting that the nonspecific binding of both tracers is similar. 11C-PBR28 was more sensitive than (R)-11C-PK11195 in the detection of TSPO overexpression in the HSE rat model, because more brain regions with significantly increased tracer uptake could be found, irrespective of the data analysis method used. These results suggest that 11C-PBR28 should be able to detect more subtle changes in microglial activation in preclinical models of neuroinflammation.

Evaluation of 4′-[Methyl-11C]Thiothymidine in a Rodent Tumor and Inflammation Model

4′-[methyl-11C]thiothymidine (11C-4DST) is a novel radiopharmaceutical that can be used for tumor imaging because of its rapid incorporation into DNA as a substrate for DNA synthesis. The in vivo stability of 11C-4DST is much greater than that of natural thymidine, because of the presence of a sulfur atom in the 4′-position. Here, we evaluated the tissue kinetics and biodistribution of 11C-4DST in a rodent tumor and acute sterile inflammation model in comparison with the previously published biodistribution data of 3′-deoxy-3′-18F-fluorothymidine (18F-FLT), 18F-FDG, 11C-choline, 11C-methionine, and 2 σ-receptor ligands in the same animal model. Methods: C6 tumor cells were implanted subcutaneously into the right shoulder and turpentine (0.1 mL) was injected intramuscularly into the left hind leg of male Wistar rats 11 d and 24 h, respectively, before the scanning day. The animals were anesthetized with isoflurane, and 11C-4DST (20–50 MBq) was injected intravenously. A dynamic PET scan was performed for 60 min with either the shoulder or hind leg region in the field of view. The animals were sacrificed, and a biodistribution study was performed. Results: 11C-4DST showed the highest tumor uptake (standardized uptake value, 4.93) of all radiopharmaceuticals tested. Its tumor-to-muscle concentration ratio (12.7) was similar to that of 18F-FDG (13.2). The selectivity of 11C-4DST for tumor as compared with acute inflammation was high (37.7), comparable to that of the σ-ligand 18F-FE-SA5845 and much higher than that of 18F-FDG (3.5). Rapidly proliferating tissues (tumor and bone marrow) showed a steadily increasing uptake. In inflamed muscle, 11C-4DST showed relatively rapid washout, and tracer concentrations in inflamed and noninflamed muscle were not significantly different at intervals greater than 40 min. Competition of endogenous thymidine for 11C-4DST uptake in target tissues was negligible, in contrast to competition for 18F-FLT uptake. Thus, pretreatment of animals with thymidine phosphorylase was not required before PET with 11C-4DST. Conclusion: In our rodent model, 11C-4DST showed high tumor uptake (sensitivity) and high tumor selectivity. The different kinetics of 11C-4DST in rapidly proliferating and inflammatory tissue may allow distinction between tumor and acute inflammation in a clinical setting. These promising results for 11C-4DST warrant further investigation in PET studies in patients with various types of tumors.

Small-Animal PET with a σ-Ligand, 11C-SA4503, Detects Spontaneous Pituitary Tumors in Aged Rats

Pituitary tumors are often detected only after death or at late stages of the disease when they are macroadenomas with a low surgical cure rate. Spontaneous pituitary tumors occur in rats over 1 y of age. In an ongoing study of changes in σ-1 agonist binding related to aging, several of our rats developed such tumors. The aim of the current study was to assess the kinetics of 11C-SA4503 (11C-labeled 1-[2-(3,4-dimethoxyphenthyl)]-4-(3-phenylpropyl)-piperazine dihydrochloride) in tumor and brain and to evaluate the utility of this tracer in the detection of pituitary tumors. Methods: Small-animal PET scans of the brain region of male Wistar Hannover rats (age, 18–32 mo) were acquired using the σ-1 agonist tracer 11C-SA4503. The time-dependent uptake of 11C in the entire brain, tumor or normal pituitary, and thyroid was measured. A 2-tissue-compartment model was fitted to the PET data, using metabolite-corrected plasma radioactivity as the input function. Results: Pituitary tumors showed up as bright hot spots in the scans. The total distribution volume (VT) of the tracer was significantly higher in the tumor than in the normal pituitary. Surprisingly, a higher VT was also seen in the brain and thyroid tissue of animals with pituitary tumors than in healthy rats. The increase in VT in the brain and thyroid was not related to a change in nondisplaceable binding potential (BPND) but rather to an increase in the partition coefficient (K1/k2) of 11C-SA4503. The increase in VT in the tumor on the other hand was accompanied by a significant increase in BPND. Western blotting analysis indicated that pituitary tumors overexpressed σ-1 receptors. Conclusion: The overexpression of σ-1 receptors in spontaneous pituitary tumors is detected as an increase in uptake and BPND of 11C-SA4503. Therefore, this tracer may have promise for the detection of pituitary adenomas, using PET.

Population pharmacokinetics of cutamesine in rats using NONMEM, C-11-SA4503, and microPET

C. Asferg, R. Møgelvang, A. Flyvbjerg, A. Frystyk, J.S. Jensen, J.L. Marott, M. Appleyard, P. Schnoh, G.B. Jensen, J. Jeppesen. Copenhagen University Hospital Glostrup, Department of Clinical Physiology and Nuclear Medicine, Glostrup, Denmark, Copenhagen University Hospital Gentofte, Department of Cardiology, Gentofte, Denmark, Aarhus University Hospital Aarhus, Medical Research Laboratories, Clinical Institute and Medical Department M, Aarhus, Denmark, Copenhagen University Hospital Bispebjerg, The Copenhagen City Heart Study, Bispebjerg, Denmark, Copenhagen University Hospital Hvidovre, Department of Cardiology, Hvidovre, Denmark, Copenhagen University Hospital Glostrup, Department of Medicine, Glostrup, Denmark, Faculty of Health Sciences, University of Aarhus, Aarhus, Denmark, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark

PET Imaging of Disease Progression and Treatment Effects in the Experimental Autoimmune Encephalomyelitis Rat Model

The experimental autoimmune encephalomyelitis model is a model of multiple sclerosis that closely mimics the disease characteristics in humans. The main hallmarks of multiple sclerosis are neuroinflammation (microglia activation, monocyte invasion, and T-cell infiltration) and demyelination. PET imaging may be a useful noninvasive technique for monitoring disease progression and drug treatment efficacy in vivo. Methods: Experimental autoimmune encephalomyelitis was induced by myelin-oligodendrocyte glycoprotein immunization in female Dark Agouti rats. Experimental autoimmune encephalomyelitis rats were imaged at baseline and at days 6, 11, 15, and 19 after immunization to monitor monocyte and microglia activation (11C-PK11195) and demyelination (11C-MeDAS) during normal disease progression and during treatment with dexamethasone. Results: 11C-PK11195 PET detected activation of microglia and monocytes in the brain stem and spinal cord during disease progression. The uptake of 11C-PK11195 was elevated in dexamethasone-treated animals that had shown mild clinical symptoms that had resolved at the time of imaging. Demyelination was not detected by 11C-MeDAS PET, probably because of the small size of the lesions (average, 0.13 mm). Conclusion: PET imaging of neuroinflammation can be used to monitor disease progression and the consequences of treatment in the experimental autoimmune encephalomyelitis rat model. PET imaging was more sensitive than clinical symptoms for detecting inflammatory changes in the central nervous system.