Anke Stahlschmidt

Longitudinal PET-MRI reveals [beta]-amyloid deposition and rCBF dynamics and connects vascular amyloidosis to quantitative loss of perfusion

The dynamics of β-amyloid deposition and related second-order physiological effects, such as regional cerebral blood flow (rCBF), are key factors for a deeper understanding of Alzheimers disease (AD). We present longitudinal in vivo data on the dynamics of β-amyloid deposition and the decline of rCBF in two different amyloid precursor protein (APP) transgenic mouse models of AD. Using a multiparametric positron emission tomography and magnetic resonance imaging approach, we demonstrate that in the presence of cerebral β-amyloid angiopathy (CAA), β-amyloid deposition is accompanied by a decline of rCBF. Loss of perfusion correlates with the growth of β-amyloid plaque burden but is not related to the number of CAA-induced microhemorrhages. However, in a mouse model of parenchymal β-amyloidosis and negligible CAA, rCBF is unchanged. Because synaptically driven spontaneous network activity is similar in both transgenic mouse strains, we conclude that the disease-related decline of rCBF is caused by CAA.

Radioiodination of 1-(2-deoxy-β-d-ribofuranosyl)-2,4-difluoro-5-iodobenzene (dRFIB), a putative thymidine mimic nucleoside for cell proliferation studies

Iodine-124 was produced via the (124)Te(p,n)(124)I reaction by 15 MeV proton irradiation of an in-house solid mass tellurium dioxide target, using the Tübingen PETtrace (General Electric Medical Systems) cyclotron. 1-(2-Deoxy-beta-D-ribofuranosyl)-2,4-difluoro-5-iodobenzene (dRFIB), a stable, non-polar thymidine mimic nucleoside, was synthesized in 5 steps following a literature method, for radioiodination with [(124)I] iodide via isotope exchange in the presence of copper sulphate and ammonium sulphate in methanol-water. The radiolabelling procedure was optimized with respect to temperature, amount of dRFIB, amount of sodium hydroxide and reaction time, to produce radiochemical yields of up to 85% with a 1-h reaction at 140 degrees C. With routine I-124 production of 30 MBq/run, relatively high specific activities, approaching 100 MBq/mmol, can be expected. The activation energy for dRFIB radioiodination was calculated from temperature-time RCY data to be approximately 100 kJ/mol using no-carrier-added [(124)I]iodide.

In Vivo Evaluation of [11C]DASB for Quantitative SERT Imaging in Rats and Mice

Serotonin, or 5-hydroxytryptamine (5-HT), plays a key role in the central nervous system and is involved in many essential neurologic processes such as mood, social behavior, and sleep. The serotonin transporter ligand 11C-3-amino-4(2-dimethylaminomethyl-phenylsufanyl)-benzonitrile (11C-DASB) has been used to determine nondisplaceable binding potential (BPND), which is defined as the quotient of the available receptor density (Bavail) and the apparent equilibrium dissociation rate constant (1/appKD) under in vivo conditions. Because of the increasing number of animal models of human diseases, there is a pressing need to evaluate the applicability of 11C-DASB to rats and mice. Here, we assessed the feasibility of using 11C-DASB for quantification of serotonin transporter (SERT) density and affinity in vivo in rats and mice. Methods: Rats and mice underwent 4 PET scans with increasing doses of the unlabeled ligand to calculate Bavail and appKD using the multiple-ligand concentration transporter assay. An additional PET scan was performed to calculate test–retest reproducibility and reliability. BPND was calculated using the simplified reference tissue model, and the results for different reference regions were compared. Results: Displaceable binding of 11C-DASB was found in all brain regions of both rats and mice, with the highest binding being in the thalamus and the lowest in the cerebellum. In rats, displaceable binding was largely reduced in the cerebellar cortex, which in mice was spatially indistinguishable from cerebellar white matter. Use of the cerebellum with fully saturated binding sites as the reference region did not lead to reliable results. Test–retest reproducibility in the thalamus was more than 90% in both mice and rats. In rats, Bavail, appKD, and ED50 were 3.9 ± 0.4 pmol/mL, 2.2 ± 0.4 nM, and 12.0 ± 2.6 nmol/kg, respectively, whereas analysis of the mouse measurements resulted in inaccurate fits due to the high injected tracer mass. Conclusion: Our data showed that in rats, 11C-DASB can be used to quantify SERT density with good reproducibility. BPND agreed with the distribution of SERT in the rat brain. It remains difficult to estimate quantitative parameters accurately from mouse measurements because of the high injected tracer mass and underestimation of the binding parameters due to high displaceable binding in the reference region.

Comparison of different Scatchard approaches for quantitative [11C]raclopride PET imaging in Mice

The increasing use of genetically engineered mice in biomedical research and the latest advances in imaging technologies provide a great potential to study receptor expression and gene function non invasively in mice. [C]raclopride (RAC) is a widely used positron emission tomography (PET) tracer to measure striatal D2 receptor binding. To determine receptor density Bmax and the apparent affinity Kd separately, multiple injections with high and low specific activities (SA) or single injections with low SA have been performed in PET studies of humans, non human primates and rats. We tested the feasibility of three approaches for D2 receptor quantification in mice: the multiple ligand concentration receptor assay (MLCRA), the transient/peak equilibrium approach (PEA) and the partial saturation approach (PSA). Since these methods differ in terms of accuracy and complexity, we aimed to identify the method, which is best suited for quantitative PET analysis in mice. 12 mice underwent a total of 3 scans with decreasing SA. Injected activity was 458±33 MBq/kg and SA ranged from 190 to 1.8 GBq/μmol, corresponding to injectedmasses of 0.02 to 0.5 μg. In sixmice the bolus injection protocol was used and receptor binding parameters were estimated from theequilibriumratio betweenbound (B) to free (F) ligand,whichwas calculated (i) from the tissue input Logangraphical approach, (ii) fromthe ratio method, (iii) at peak equilibrium and (iv) at partial saturation. Receptor occupancy plotted as a function of log10(1/SA). For the PSA an injected mass of 4.5 μg was chosen. In addition, we used the bolus plus constant infusion protocol (BI) to attain true equilibrium between bound and free tracer concentrations, avoiding difficult arterial cannulation in mice. Six mice underwent three 90 min emission scans using a Kbol of 88 min. The tracer concentration was adjusted to 148 MBq/ml. B/F was calculated (i) from the Logan graphical analysis and (ii) from the ratio method. The average D2 receptor density Bmax was 26±4 pmol/ml and the apparent Kd was 13±2 pmol/ml using the bolus plus constant infusion protocol, which was defined as gold standard. If the tracer was injected by bolus, we found lower values for Bmax and Kd when using the Logan graphical analysis (Bmax=15±4; Kd=7±1) and the ratio from last 30 min (Bmax=22±4; Kd=10±2) and higher values when using the PEA (Bmax=46±4; Kd=23±2) and the PSA (Bmax=35±8; Kd=15±6). The receptor occupancy curves showed that an injected tracer mass of 0.07 μg induces approximately 10% receptor occupancy with corresponding SA values of 1400 Ci/mmol. Our data showed that the tracer mass, if higher than 0.07 μg can highly effect binding parameter estimations and has to be taken into account when performing kinetic analysis, specifically in mice. We also demonstrated that in vivo determination of D2 receptor density and affinity using multiple injection protocols and single injection protocols is feasible in mice. However slight overand underestimations were found when comparing the different analysis methods to the bolus plus constant infusion protocol.

CycloSal-dRFIB, a Thymidine Mimetic, Thymidine Kinase by-Pass Nucleoside Prodrug: Radioiodination, in vitro Cellular Uptake and Biodistribution in Murine Models

Cyclo-(3-methylsaligenyl)-5-O-[1-(2,4-difluoro-5-[125I]iodophenyl)-2-deoxy-β-D-ribofuranosyl]phosphate (cycloSal- dRF[125I]IB) was radioiodinated with sodium [125I]iodide via copper-catalyzed isotope exchange in 48% radiochemical yield. cycloSal-dRF[125I]IB was found to be incorporated into the cytoplasmic nucleic acid and mitochondrial fractions of murine KBALB and K-STK (engineered to express HSV-1 thymidine kinase) cells in cell culture. Uptake was greater than that for either the corresponding nucleoside dRF[125I]IB or [125I]IUdR. These in vitro studies support a mechanism of metabolic activation to the free nucleotide, thereby effecting TK-bypass. Pharmacokinetic studies in rats reflect a complex interplay of tissue depot effects, hepatobiliary recycling, and metabolism. Biodistribution studies in tumor- bearing mice provide further evidence for lipophilic depot effects and hepatobiliary recirculation, with no evidence for active (metabolic) accumulation in any tissue.

Biodistribution and Imaging of 1-(2-Deoxy-β-D-Ribofuranosyl)-2,4-Difluoro-5-[123/125I]Iodobenzene (dRF[123/125I]IB), A Nonpolar Thymidine-Mimetic Nucleoside, in Rats and Tumor-Bearing Mice

1-(2-Deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-iodobenzene (dRFIB) is a putative bioisostere of iododeoxyuridine (IUdR). The advantages of dRFIB over IUdR for in vivo studies include resistance to both phosphorolytic cleavage of the nucleoside bond and de-iodination. dRFIB was radioiodinated (dRF123/125IB) by copper-catalyzed exchange using commercial sodium [123/125I]iodide. The in vivo biodistribution of dRF[125I]IB in BALBc mice and imaging of dRF[123I]IB in Sprague-Dawley rats are reported. In vivo data for rats show rapid clearance of radioactivity from blood (>95%ID in 15 minutes), extensive excretion in urine (56%ID/24 hours), concentration in the hepatobiliary-small intestine system and very little fecal excretion (∼3%ID/24 hours). Pharmacokinetic data for dRF[125I]IB (i.v. 48.7 ug/kg) in rats (t1/2[h] = 0.51 ± 0.14, AUCinf[μg.min/mL] = 3.7 ± 0.4, Cl[L/kg/h] = 0.75 ± 0.12, Vss[L/kg] = 0.96 ± 0.18) confirm previously reported dose-dependent pharmacokinetics. Scintigraphic images of rats dosed with dRF[123I]I were compatible with rapid soft-tissue clearance and extensive accumulation of radioactivity in bladder/urine and liver/small intestine. In tumor-bearing mice, thyroid and stomach radioactivity was indicative of moderate deiodination. An unidentified polar radioactive metabolite was detected in serum.