Emmanuelle Briard

We describe the identification of a candidate positron emission tomography (PET) imaging agent for the NLRP3 protein. NLRP3 plays a critical role in the immune system and has proven a difficult target for the development of imaging agents due to its low and cell‐specific expression profile. A recently described series of pyridazine‐based inhibitors, with improved permeability and brain‐penetration properties, was used as a starting point for the development of a suitable PET imaging agent. Optimization of affinity, non‐specific binding and pharmacokinetic properties led to the identification of aminopyridazine (R)‐2‐(6‐((1‐cyclopropylpiperidin‐3‐yl)amino)pyridazin‐3‐yl)‐5‐fluoro‐3‐methylphenol (17 b), which meets the preclinical profile of a successful imaging agent, and whose tritiated version demonstrated excellent specificity in a radioligand saturation binding assay, confirming its imaging potential.¹⁸F labeling led to [¹⁸F]NP3‐627, the proposed PET imaging agent.

Nonspecific binding (NSB) is a key parameter in optimizing PET imaging tracers. We compared the ability to predict NSB of three available methods: LIMBA, rat f_u,brain, and CHI(IAM). Even though NSB is often associated with lipophilicity, we observed that logD does not correlate with any of these assays, clearly indicating that lipophilicity, while influencing NSB, is insufficient to predict it. A cross‐comparison of the methods showed that all three correlate and are useful predictors of NSB. The three assays, however, rank the molecules slightly differently, illustrating the challenge of comparing molecules within a narrow chemical space. We also noted that CHI(IAM) values more effectively predict V_NS, a measure of in vivo NSB in the human brain. CHI(IAM) measurements might be a closer model of the actual physicochemical interaction between PET tracer candidates and cell membranes, and seems to be the method of choice for the optimization of in vivo NSB.

Autotaxin (ATX) is a secreted enzyme with tissue levels associated with tissue injury, which increase during wound healing and chronic fibrotic diseases. We selected [¹⁸F](R,E)‐3‐(4‐chloro‐2‐((5‐methyl‐2H‐tetrazol‐2‐yl)methyl)phenyl)‐1‐(4‐((5‐(2‐fluoroethoxy)pyridin‐2‐yl)methyl)‐2‐methylpiperazin‐1‐yl)prop‐2‐en‐1‐one ([¹⁸F]PRIMATX, [¹⁸F]2), a tracer for positron emission tomography, to image ATX expression in vivo. It successfully differentiates expression levels in lung tissue samples from idiopathic pulmonary fibrosis patients, and allows the detection of ATX‐expressing tumors in living mice, confirming its potential for development as a clinical imaging agent.

Imaging T cells using positron emission tomography (PET) would be highly useful for diagnosis and monitoring in immunology and oncology patients. There are, however, no obvious targets that can be used to develop imaging agents for this purpose. We evaluated several potential target proteins with selective expression in T cells, and for which lead molecules were available: protein kinase C isozyme θ (PKC θ), lymphocyte‐specific protein tyrosine kinase (Lck), zeta‐chain‐associated protein kinase 70 (ZAP70), and interleukin‐2‐inducible T‐cell kinase (Itk). Ultimately, we focused on Itk and identified a tool molecule with properties suitable for in vivo imaging of T cells: (5aR)‐5,5‐difluoro‐5a‐methyl‐N‐(1‐((S)‐3‐(methylsulfonyl)phenyl)(tetrahydro‐2H‐pyran‐4‐yl)methyl)‐1H‐pyrazol‐4‐yl)‐1,4,4a,5,5a,6‐hexahydrocyclopropa[f]indazole‐3‐carboxamide (23). Although it does not have the optimal profile for clinical use, this molecule indicates that it might be possible to develop Itk‐selective PET ligands for imaging the distribution of T cells in patients.

BAF312 (siponimod) is a sphingosine‐1‐phosphate (S1P) receptor modulator in clinical development for the treatment of multiple sclerosis, with faster organ/tissue distribution and elimination kinetics than its precursor FTY720 (fingolimod). Our aim was to develop a tracer to better quantify the penetration of BAF312 in the human brain, with the potential to be labeled for positron emission tomography (PET) or single‐photon emission computed tomography (SPECT). Although the PET radioisotopes ¹¹C and ¹⁸F could have been introduced in BAF312 without modifying its structure, they do not have decay kinetics compatible with the time required for observing the drug′s organ distribution in patients. In contrast, the SPECT radioisotope ¹²³I has a longer half‐life and would suit this purpose. Herein we report the identification of an iodinated derivative of BAF312, (E)‐1‐(4‐(1‐(((4‐cyclohexyl‐3‐iodobenzyl)oxy)imino)ethyl)‐2‐ethylbenzyl)azetidine‐3‐carboxylic acid (18, MS565), as a SPECT tracer candidate with affinity, S1P receptor selectivity, overall physicochemical properties, and blood pharmacokinetics similar to those of the original molecule. A whole‐body autoradiography study performed with [¹⁴C]MS565 subsequently confirmed that its organ distribution is similar to that of BAF312. This validates the selection of MS565 for ¹²³I radiolabeling and for use in imaging studies to quantify the brain penetration of BAF312.

Orexin receptor antagonists represent attractive targets for the development of drugs for the treatment of insomnia. Both efficacy and safety are crucial in clinical settings and thorough investigations of pharmacokinetics and pharmacodynamics can predict contributing factors such as duration of action and undesirable effects. To this end, we studied the interactions between various “dual” orexin receptor antagonists and the orexin receptors, OX₁R and OX₂R, over time using saturation and competition radioligand binding with [³H]-BBAC ((S)-N-([1,1′-biphenyl]-2-yl)-1-(2-((1-methyl-1H-benzo[d]imidazol-2-yl)thio)acetyl)pyrrolidine-2-carboxamide). In addition, the kinetics of these compounds were investigated in cells expressing human, mouse and rat OX₁R and OX₂R using FLIPR^® assays for calcium accumulation. We demonstrate that almorexant reaches equilibrium very slowly at OX₂R, whereas SB-649868, suvorexant, and filorexant may take hours to reach steady state at both orexin receptors. By contrast, compounds such as BBAC or the selective OX₂R antagonist IPSU ((2-((1H-Indol-3-yl)methyl)-9-(4-methoxypyrimidin-2-yl)-2,9-diazaspiro[5.5]undecan-1-one) bind rapidly and reach equilibrium very quickly in binding and/or functional assays. Overall, the “dual” antagonists tested here tend to be rather unselective under non-equilibrium conditions and reach equilibrium very slowly. Once equilibrium is reached, each ligand demonstrates a selectivity profile that is however, distinct from the non-equilibrium condition. The slow kinetics of the “dual” antagonists tested suggest that in vitro receptor occupancy may be longer lasting than would be predicted. This raises questions as to whether pharmacokinetic studies measuring plasma or brain levels of these antagonists are accurate reflections of receptor occupancy in vivo.

FTY720 (fingolimod, Gilenya®) is a sphingosine 1‐phosphate (S1P) receptor modulator that shows significant therapeutic efficacy after oral administration to patients of multiple sclerosis. Because FTY720 does not contain any atom whose PET or SPECT radioisotope would have a half‐life compatible with its pharmacokinetic properties, it cannot be used directly for imaging. Instead, we propose BZM055 as a surrogate tracer to study its pharmacokinetics and organ distribution in patients and, given that FTY720 accumulates in myelin sheaths, for myelin imaging. BZM055 (2 a, 2‐iodo‐FTY720) can be easily radiolabeled with ¹²³I (for SPECT) or ¹²⁴I (for PET). Not only does it closely mimic the pharmacokinetics and organ distribution of FTY720, but also its affinity, selectivity for S1P receptors, phosphorylation kinetics, and overall physicochemical properties. [¹²³I]BZM055 is currently under development for clinical imaging.