Luca Gobbi

Biochemical and behavioural characterization of EMPA, a novel high‐affinity, selective antagonist for the OX2 receptor

Background and purpose:  The OX2 receptor is a G‐protein‐coupled receptor that is abundantly found in the tuberomammillary nucleus, an important site for the regulation of the sleep‐wake state. Herein, we describe the in vitro and in vivo properties of a selective OX2 receptor antagonist, N‐ethyl‐2‐[(6‐methoxy‐pyridin‐3‐yl)‐(toluene‐2‐sulphonyl)‐amino]‐N‐pyridin‐3‐ylmethyl‐acetamide (EMPA).

Discovery of a High Affinity and Selective Pyridine Analog as a Potential Positron Emission Tomography Imaging Agent for Cannabinoid Type 2 Receptor

As part of our efforts to develop CB2 PET imaging agents, we investigated 2,5,6-substituted pyridines as a novel class of potential CB2 PET ligands. A total of 21 novel compounds were designed, synthesized, and evaluated for their potency and binding properties toward human and rodent CB1 and CB2. The most promising ligand 6a was radiolabeled with carbon-11 to yield 16 ([(11)C]RSR-056). Specific binding of 16 to CB2-positive spleen tissue of rats and mice was demonstrated by in vitro autogadiography and verified in vivo in PET and biodistribution experiments. Furthermore, 16 was evaluated in a lipopolysaccharid (LPS) induced murine model of neuroinflammation. Brain radioactivity was strikingly higher in the LPS-treated mice than the control mice. Compound 16 is a promising radiotracer for imaging CB2 in rodents. It might serve as a tool for the investigation of CB2 receptor expression levels in healthy tissues and different neuroinflammatory disorders in humans.

Radioligand development for molecular imaging of the central nervous system with positron emission tomography

Positron emission tomography (PET) is routinely used to support the development of drugs to treat neurological and psychiatric disorders. PET radioligands must not only be selective for the target of interest but must also possess a range of physicochemical and pharmacological characteristics that allow them to be radiolabelled with short-lived positron-emitting isotopes, safely administered to humans, and for the degree of target binding to be quantified in vivo. We review the ligand development process, including target selection, radioligand discovery (in vitro and preclinical evaluation), radiochemistry and evaluation in humans.

Total syntheses of multiple cladiellin natural products by use of a completely general strategy.

The enantioselective total syntheses of 10 cladiellin natural products have been completed, starting from the known allylic alcohol (+)-14, which can be prepared in large quantities. The bridged tricyclic core of the cladiellins has been constructed via three ring-forming reactions: (i) an intramolecular reductive cyclization between an aldehyde and an unsaturated ester, mediated by samarium(II) iodide, to form a tetrahydropyranol; (ii) reaction of a metal carbenoid, generated from a diazo ketone, with an ether to produce an ylide-like intermediate that rearranges to produce E- or Z-oxabicyclo[6.2.1]-5-undecen-9-one; and (iii) a Diels-Alder cycloaddition reaction to construct the third ring found in the core structure of the cladiellins. The key ring-forming reaction, in which a diazo ketone is converted into a bridged bicyclic ether, can be tuned to give either of the isomeric oxabicyclo[6.2.1]-5-undecen-9-ones as the major product by switching from a copper to a rhodium catalyst and selecting the appropriate reaction conditions. The tricyclic products obtained from the three-step sequence involving the Diels-Alder cycloaddition reaction can be employed as advanced intermediates to prepare a wide range of cladiellin natural products.

Label-free assay for the assessment of nonspecific binding of positron emission tomography tracer candidates.

Positron emission tomography (PET) is a valuable non-invasive technique for the visualization of drug tissue distribution and receptor occupancy at the target site in living animals and men. Many potential PET tracers, however, fail due to an unfavorably high non-specific binding (NSB) to non-target proteins and phospholipid membranes which compromises the sensitivity of PET. Hence, there is a high demand to assess the extent of NSB as early as possible in the PET tracer development process, preferentially before ligands are radiolabeled and elaborate imaging studies are performed. The purpose of this study was to establish a novel Lipid Membrane Binding Assay (LIMBA) for assessing the tendency of potential tracers to bind non-specifically to brain tissue. The assay works with unlabeled compounds and allows the medium-throughput measurement of brain tissue/water distribution coefficients, logDbrain (pH7.4), at minimal expense of animal tissue. To validate LIMBA, logDbrain (pH7.4) values were measured and compared with NSB estimates derived from in vivo PET studies in human brain (n=10 tracers, literature data), and in vitro autoradiography studies in rat and mouse brain slices (n=30 tritiated radioligands). Good agreement between logDbrain (pH7.4) and the volume of distribution in brain of non-specifically bound tracer in PET was achieved, pertaining to compounds classified as non-substrates of P-glycoprotein (R(2)≥0.88). The ability of LIMBA for the prediction of NSB was further supported by the strong correlation between logDbrain (pH7.4) and NSB in brain autoradiography (R(2)≥0.76), whereas octanol/water distribution coefficients, logDoct (pH7.4) were less predictive. In conclusion, LIMBA provides a fast and reliable tool for identifying compounds with unfavorably high NSB in brain tissue. The data may be used in conjunction with other parameters like target affinity, density and membrane permeability for the selection of most promising compounds to be further investigated in vivo as potential novel PET tracers.

Preclinical Evaluation of 18F-RO6958948, 11C-RO6931643, and 11C-RO6924963 as Novel PET Radiotracers for Imaging Tau Aggregates in Alzheimer Disease

Tau aggregates and amyloid-β (Aβ) plaques are key histopathologic features in Alzheimer disease (AD) and are considered targets for therapeutic intervention as well as biomarkers for diagnostic in vivo imaging agents. This article describes the preclinical in vitro and in vivo characterization of 3 novel compounds—RO6958948, RO6931643, and RO6924963—that bind specifically to tau aggregates and have the potential to become PET tracers for future human use. Methods: RO6958948, RO6931643, and RO6924963 were identified as high-affinity competitors at the 3H-T808 binding site on native tau aggregates in human late-stage AD brain tissue. Binding of tritiated compounds to brain tissue sections of AD patients and healthy controls was analyzed by macro- and microautoradiography and by costaining of tau aggregates and Aβ plaques on the same tissue section using specific antibodies. All 3 tracer candidates were radiolabeled with a PET nuclide and tested in vivo in tau-naïve baboons to assess brain uptake, distribution, clearance, and metabolism. Results: 3H-RO6958948, 3H-RO6931643, and 3H-RO6924963 bound with high affinity and specificity to tau aggregates, clearly lacking affinity for concomitant Aβ plaques in human AD Braak V tissue sections. The specificity of all 3 radioligands for tau aggregates was supported, first, by binding patterns in AD sections comparable to the tau-specific radioligand 3H-T808; second, by very low nonspecific binding in brain tissue devoid of tau pathology, excluding significant radioligand binding to any other central nervous system target; and third, by macroscopic and microscopic colocalization and quantitative correlation of radioligand binding and tau antibody staining on the same tissue section. RO6958948, RO6931643, and RO6924963 were successfully radiolabeled with a PET nuclide at high specific activity, radiochemical purity, and yield. After intravenous administration of 18F-RO6958948, 11C-RO6931643, and 11C-RO6924963 to baboons, PET scans indicated good brain entry, rapid washout, and a favorable metabolism pattern. Conclusion: 18F-RO6958948, 11C-RO6931643, and 11C-RO6924963 are promising PET tracers for visualization of tau aggregates in AD. Head-to-head comparison and validation of these tracer candidates in AD patients and healthy controls will be reported in due course.

First in-human PET study of 3 novel tau radiopharmaceuticals: [11C]RO6924963, [11C]RO6931643, and [18F]RO6958948

11C-RO-963, 11C-RO-643, and 18F-RO-948 (previously referred to as 11C-RO6924963, 11C-RO6931643, and 18F-RO6958948, respectively) have been reported as promising PET tracers for tau imaging based on in vitro and preclinical PET data. Here we describe the first, to our knowledge, human evaluation of these novel radiotracers. Methods: Amyloid PET–positive Alzheimer disease (AD) subjects and younger controls each received 2 different tau tracers. Dynamic 90-min scans were obtained after bolus injection of 11C-RO-963, 11C-RO-643, or 18F-RO-948. Arterial blood sampling was performed on 11 healthy controls and 11 AD subjects. Regions were defined on MR images, and PET data were quantified by plasma reference graphical analysis (for total distribution volume) and target cerebellum ratio (SUV ratios of 60- to 90-min frames). SUV ratio images were also analyzed voxelwise. Five older controls each underwent 2 scans with 18F-RO-948 for evaluation of test–retest variability. Four AD subjects underwent a repeated 18F-RO-948 scan 6–22 mo after the first scan. Six additional healthy controls (3 men and 3 women; age range, 41–67 y) each underwent 1 whole-body dosimetry scan with 18F-RO-948. Results: In younger controls, SUVpeak was observed in the temporal lobe with values of approximately 3.0 for 11C-RO-963, 1.5 for 11C-RO-643, and 3.5 for 18F-RO-948. Over all brain regions and subjects, the trend was for 18F-RO-948 to have the highest SUVpeak, followed by 11C-RO-963 and then 11C-RO-643. Regional analysis of SUV ratio and total distribution volume for 11C-RO-643 and 18F-RO-948 clearly discriminated the AD group from the healthy control groups. Compartmental modeling confirmed that 11C-RO-643 had lower brain entry than either 11C-RO-963 or 18F-RO-948 and that 18F-RO-948 showed better contrast between (predicted) areas of high versus low tau accumulation. Thus, our subsequent analysis focused on 18F-RO-948. Both voxelwise and region-based analysis of 18F-RO-948 binding in healthy controls versus AD subjects revealed multiple areas where AD subjects significantly differed from healthy controls. Of 22 high-binding regions, 13 showed a significant group difference (after ANOVA, F(1,21) = 45, P < 10−5). Voxelwise analysis also revealed a set of symmetric clusters where AD subjects had higher binding than healthy controls (threshold of P < 0.001, cluster size > 50). Conclusion: 18F-RO-948 demonstrates characteristics superior to 11C-RO-643 and 11C-RO-963 for characterization of tau pathology in AD. Regional binding data and kinetic properties of 18F-RO-948 compare favorably with other existing tau PET tracers.

Preparation of [18F]-N-(2-fluoro-ethyl)-N-methylamine as a building block for PET radiopharmaceuticals.

We have investigated the use of cyclic sulfamidates as precursors to yield secondary amines as building blocks for subsequent reaction with carboxylic acids and acyl chlorides. The preparation of the protonated form of [(18)F]-N-(2-fluoro-ethyl)-N-methylamine from the corresponding cyclic sulfamidate proceeded within a one pot two-step procedure (81 ± 12%, n = 10). The secondary amine reacted readily with acyl chlorides and/or carboxylic acids giving amides with yields ranging from 4 to 17% at the end of synthesis (182 ± 12 min). The new methodology provides a practical approach for the labelling of molecules where intramolecular cyclisation of precursors is favoured under typical radiofluorination conditions.

ON EVALUATION OF TAU ACCUMULATIONS IN LONGITUDINAL STUDIES OF ALZHEIMER’S DISEASE (AD): IMPLICATIONS FROM A PET STUDY WITH [18F]RO6958948

A1 19 A2 8 A3 22 A4 21 ACCUMULATIONS IN LONGITUDINAL STUDIES OFALZHEIMER’S DISEASE (AD): IMPLICATIONS FROMAPET STUDYWITH [18F]RO6958948 Hiroto Kuwabara, Edilio Borroni, Robert A. Comley, Joshua Roberts, Kelly Kitzmiller, Paul B. Rosenberg, Madhav Thambisetty, Michael Honer, Gregory Klein, Lorena Gapasin, Luca Gobbi, Dean F. Wong, Johns Hopkins University School of Medicine, Baltimore, MD, USA; Roche Pharmaceutical Research and Early Development, Basel, Switzerland; Abbvie Inc., Chicago, IL, USA. Contact e-mail: hkuwaba1@jhmi.edu

Distribution of BACE1 in the rodent, monkey and human brain

fully created a method to measure hippocampus volumes in live mice and serves as a first step toward understanding a possible biomarker for the disease. Methods: An in-vivo T 2 -weighted imaging method was developed with optimal contrast and resolution to allow for straightforward segmentation and volume determination of the hippocampus in mice. 7-month old, single transgenic mice, expressing either a chimeric mouse/human amyloid precursor protein (APP) mutation or a mutant human presenilin 1 (PS1) were imaged multiple times using 3D T 2 -weighted imaging on a 7T magnet (Figure 1) to establish the method and determine its accuracy. To calculate the volume, the hippocampus of each mouse was both manually segmented by three individuals and segmented with a semi-automated method (Figure 2). The images were also registered using an affine transformation to determine shape changes. Results: The hippocampus volumes ranged from 18 mm 3 to 23 mm 3 when done by the manual segmentation method. The semi-automated method gave 30% larger volumes as compared to the manual method (Figure 3). The kappa index, a measure of overlap between manually and automatically segmented brain structures, had values of > 0.86 for each compared image. The registration data showed that there were no significant shape changes for any of the hippocampi. Conclusions:A reliable method able to detect 1 mm 3 volume measurements when performed by a single individual was developed. The semi-automated segmentation was unable to detect differences. These results suggest that manual segmentation is still considered the most reliable segmentation method for small structures.