Evaluation of specific binding of [(11)C]TZ7774 to the receptor-interacting protein kinase 1 (RIPK1) in the brain.

Am J Nucl Med Mol Imaging 2024;14(5):345-350 www.ajnmmi.us /ISSN:2160-8407/ajnmmi0157990 Original Article Evaluation of specific binding of [11C]TZ7774 to the receptor-interacting protein kinase 1 (RIPK1) in the brain Takayuki Sakai1, Takashi Yamada1,2, Hiroshi Ikenuma1, Aya Ogata1,3, Masanori Ichise1, Saori Hattori1, Junichiro Abe1, Mari Tada4, Akiyoshi Kakita4, Masaaki Suzuki1, Kengo Ito1, Takashi Kato1, Shinichi Imamura1, Yasuyuki Kimura1 Department of Clinical and Experimental Neuroimaging, Center for Development of Advanced Medicine for Dementia, National Center for Geriatrics and Gerontology, Obu, Aichi, Japan; 2Department of Human Life and Sciences Division of Health and Nutritional Science, Nagoya University of Economics, Nagoya, Japan; 3Department of Pharmacy, Faculty of Pharmacy, Gifu University of Medical Science, Kani, Japan; 4Department of Pathology, Brain Research Institute, Niigata University, Niigata, Japan 1 Received May 23, 2024; Accepted August 25, 2024; Epub October 15, 2024; Published October 30, 2024 Abstract: Microglia, a type of immune cells of the central nervous system, play a critical role in the pathophysiology of neurodegenerative disorders including Alzheimer’s disease (AD). Recently, efforts for drug discovery have focused on modifying the function of microglia to halt AD progression. One such effort targets a multifaceted kinase called receptor-interacting protein kinase 1 (RIPK1) that controls inflammation and cell death. Pharmaceutical inhibition of RIPK1 in microglia prevents their homeostatic status from transforming to disease-associated status. Thus, RIPK1 inhibitors can be a therapeutic agent for halting AD progression. Therefore, in vivo imaging of RIPK1 may be a useful biomarker of AD. Recently, a novel PET ligand, [11C]TZ7774, targeting RIPK1 was developed showing its ability to enter the brain and an increased uptake in the spleen of acute inflammation model mice. However, they have not yet shown direct evidence of specific binding of [11C]TZ7774 to RIPK1 in the brain. In this study, we replicated the synthesis of [11C]TZ7774 and examined its specific binding in the rat and human brain. Our studies with this ligand failed to detect sufficient specific binding of [11C]TZ7774 to RIPK1 in the brain neither by PET imaging with healthy and acute inflammation model rats, nor by autoradiography with healthy rat and human brain slices. Our results suggest that the RIPK1 ligand, [11C]TZ7774, is unlikely to be useful in humans. Future studies are warranted to develop more optimal radioligands for PET imaging of RIPK1. Keywords: Receptor-interacting protein kinase 1, Alzheimer’s disease, neuroinflammation, positron emission tomography Introduction Microglia, a type of immune cells of the central nervous system, play a critical role in the pathophysiology of neurodegenerative disorders including Alzheimer’s disease (AD). In AD, phenotypically activated microglia that govern neural immunity are closely associated with senile plaques and neurofibrillary tangles [1]. The activated microglia have a significant impact on the formation of these pathological lesions and concurrent neurodegeneration [2]. Recently, efforts of drug discovery have focused on modifying the function of microglia to halt AD progression. One such effort targets a multifaceted kinase called receptor-interacting protein kinase 1 (RIPK1) that controls inflammation and cell death [3]. Pharmaceutical inhibition of RIPK1 in microglia prevents their homeostatic status from transforming to diseaseassociated status [4]. Thus, RIPK1 inhibitors can be a therapeutic agent for halting AD progression, as well as a potential in vivo imaging biomarker of AD. To our knowledge, structurally different types of PET ligands for imaging of RIPK1 have been developed to date with high RIPK1 inhibitory activities (Figure 1). The first type of inhibitors are necrostatin (Nec-1) and Nec-1s. Necrostatin was the first RIPK1 inhibitor discovered by screening a chemical library containing 15,000 com- pounds [EC50 (Jurkat) = 494 nM] [5]. However, Nec-1 inhibits RIPK1 non-selectively due to its structural characteristics [6]. It also has low metabolic stability in vivo. Subsequent structure-activity relationship analysis discovered Nec-1s with an approximately 2.4-fold inhibitory activity of Nec-1 (EC50 = 206 nM) and improved metabolic stability [7]. The second type of inhibitor is GSK’963 [IC50(RIPK1) = 29 nM], which effectively inhibited programmed necrosis of mouse and human cells at IC50 values of 1.0 nM and 4.0 nM, respectively [8]. The third type of inhibitor is benzoxazepine, 1 (TZ7774, Figure 1), exhibits a high pharmacological activity (Ki = 0.93 nM) and selectivity for RIPK1, and is also pharmacokinetically stable against RIPK1 [9]. The three structurally distinct PET ligands developed based on these RIPK1 inhibitors have been referred to as [18F]CNY-07, [11C]GG502, and [11C]TZ7774, respectively (Figure 1, bottom structures). The first two PET ligands, [18F]CNY-07 and [11C]GG502, were noted to be taken up into the mouse brain. However, their specific binding was not clearly demonstrated [10, 11]. In the report of [11C] TZ7774, this ligand was used in mice and macaques, showing its ability to take up into the brain [12]. Their biodistribution study demonstrated that ligand uptake was increased in the spleen of LPS-induced acute inflammation model mice. These findings suggested that this ligand https://doi.org/10.62347/PAZG6300 Specific binding of [11C]TZ7774 to RIPK1 in the brain Figure 1. Structures of typical RIPK1-targeted inhibitors and PET ligands so far developed. could be used as a PET radioligand for imaging RIPK1 in vivo in animals. However, they have not shown any direct evidence of specific binding of [11C]TZ7774 to RIPK1 in the brain. In this study, we evaluated it’s specific binding to RIPK1 by PET in the rat brain and additionally by autoradiography of brain sections of rats and humans. We selected [11C]TZ7774 for testing because it has a much higher potential to exhibit specific binding to RIPK1, given its greater potency compared to the others, and no direct evidence of its specific binding has yet been demonstrated. Methods Chemicals and synthesis The precursor compound (TZ7790) and standard compound (TZ7774) were synthesized according to the method to synthesize compound 22 (TZ7774) reported by Yoshikawa et al. [9] (Scheme S1). The structure of [11C] TZ7774 was achieved by reacting TZ7790 [11C]CH3I under basic conditions [12] (Scheme S2). The structure of [11C] TZ7774 was identified by comparing its HPLC retention time to that of the standards, TZ7774 (Figure S1). Details are described in the Supplementary Information. Animals The current PET imaging study utilized male F344/NSlc rats, 7 weeks old, obtained from Japan SLC, Inc., Hamamatsu, Japan. The animals involved in this study were housed and treated according to the National Research Council’s Guide for the Car (...truncated)