Dieter Muri

Iridium‐Catalyzed H/D Exchange: Ligand Complexes with Improved Efficiency and Scope

Hydrogen isotope exchange (HIE) is one of the most attractive tools for the introduction of deuterium or tritium to an organic compound. Herein, iridium complexes with N,P-ligands, highly active catalysts for asymmetric double bond reductions, have been tested for their HIE capabilities. Electron-rich ligands, containing dicyclohexylphosphines or phosphinites, have been identified as excellent ligands for efficient deuterium incorporation. Substrates with strong directing groups, that is, pyridines, ketones, and amides, as well as weak ligating units, such as, nitro, sulfones, and sulfonamides, could be labeled efficiently. With the addition of tris(pentafluorophenyl) borane to the reaction mixture, also highly deactivating nitrile substituents were well tolerated in the reaction. Based on the excellent results obtained with the chiral ThrePhox ligand, a structurally simpler, achiral ligand was developed. The iridium complex containing this ligand, proved to be a powerful catalyst for HIE reactions.

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.

Nanotracing and cavity-ring down spectroscopy: A new ultrasensitive approach in large molecule drug disposition studies

New therapeutic biological entities such as bispecific antibodies targeting tissue or specific cell populations form an increasingly important part of the drug development portfolio. However, these biopharmaceutical agents bear the risk of extensive target-mediated drug disposition or atypical pharmacokinetic properties as compared to canonical antibodies. Pharmacokinetics and bio-distribution studies become therefore more and more important during lead optimization. Biologics present, however, greater analytical challenges than small molecule drugs due to the mass and selectivity limitation of mass spectrometry and ligand-binding assay, respectively. Radiocarbon (14C) and its detection methods, such as the emerging 14C cavity ring down spectroscopy (CRDS), thus can play an important role in the large molecule quantitation where a 14C-tag is covalently bound through a stable linker. CRDS has the advantage of a simplified sample preparation and introduction system as compared to accelerator mass spectrometry (AMS) and can be accommodated within an ordinary research laboratory. In this study, we report on the labeling of an anti-IL17 IgG1 model antibody with 14C propionate tag and its detection by CRDS using it as nanotracer (2.1 nCi or 77.7 Bq blended with the therapeutic dose) in a pharmacokinetics study in a preclinical species. We compare these data to data generated by AMS in parallel processed samples. The derived concentration time profiles for anti-IL17 by CRDS were in concordance with the ones derived by AMS and γ-counting of an 125I-labeled anti-IL17 radiotracer and were well described by a 2-compartment population pharmacokinetic model. In addition, antibody tissue distribution coefficients for anti-IL17 were determined by CRDS, which proved to be a direct and sensitive measurement of the extravascular tissue concentration of the antibody when tissue perfusion was applied. Thus, this proof-of-concept study demonstrates that trace 14C-radiolabels and CRDS are an ultrasensitive approach in (pre)clinical pharmacokinetics and bio-distribution studies of new therapeutic entities.

Tools for work-up and prepurification of tritium-labeled small molecules

This practitioner protocol describes the use and application of different types of solid-phase extractions and metal scavengers for small-scale reactions, particularly in the context of purifying tritiated molecules from heavily contaminated reaction mixtures. Polymer-bound strong cation exchangers are especially suitable for separating basic compounds from neutral or acidic molecules and have been widely applied in the work-up of iridium-based hydrogen isotope exchange reactions. Polymer-bound strong anion exchangers can help to separate acidic compounds from their neutral or basic counterparts or to easily convert a trifluoroacetate or formate salt to its free base after HPLC purification. Metal scavengers have been used for the work-up of metal-catalyzed coupling reactions and hydrogen isotope exchange reactions, facilitating subsequent chromatographic purifications.

Development of a safe and scalable route towards a tau PET tracer precursor

A scalable 5-step synthesis of the diazacarbazole derivative 1 used as tau PET tracer precursor is reported. Key features of this synthesis include a Buchwald-Hartwig amination, a Pd catalyzed CH activation and a Suzuki-Miyaura cross-coupling.

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.