ORIGINAL RESEARCH article
Front. Pharmacol.
Sec. Pharmacology of Anti-Cancer Drugs
Volume 16 - 2025 | doi: 10.3389/fphar.2025.1671065
This article is part of the Research TopicDevelopment and Validation of New Molecular Probes of Nuclear Medicine and New Targets of Nuclear Drugs in CancersView all 8 articles
Clozapine Responsible for In vivo Imaging and Pharmacological Effect of Clozapine-N-oxide (CNO) in Murine DREADD Models
Provisionally accepted
- 1Tianjin Medical University Cancer Institute and Hospital, Tianjin, China
- 2Fudan University, Shanghai, China
- 3National Institutes for Quantum Science and Technology, Chiba, Japan
- 4Tianjin Medical University, Tianjin, China
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Although clozapine-N-oxide (CNO), a major metabolite of clozapine (CLZ), is a widely used agonist of Designer Receptors Exclusively Activated by Designer Drug (DREADD), the compound responsible for visualizing or activating DREADDs in nuclear medicine imaging and at pharmacological doses in murine models remains unclear. In this study, we performed positron emission tomography (PET) imaging and ex vivo autoradiography with 11C-CNO and 11C-clozapine (11C-CLZ) to detect human M4 muscarinic acetylcholine receptor DREADD (hM4D) expression in both a viral vector-injected intracranial mouse model and an original transgenic (Tg) mouse line. PET and autoradiographic images confirmed that both 11C-CNO and 11C-CLZ enabled visualization of hM4D expression in the brain. However, metabolite analysis revealed that the brain concentration of 11C-CLZ was approximately 40 times higher than that of 11C-CNO, while its plasma concentration was only 40% of 11C-CNO at 60 minutes post-injection. In Tg and nonTg mice intraperitoneally administered a pharmacological dose of CNO (1 mg/kg), the brain ratios of non-radiolabeled CLZ to CNO ranged from 25 to 263, whereas plasma ratios ranged from 0.04 to 0.11 at 30 and 60 minutes post-injection. Notably, intraperitoneal administration of a low CLZ dose (0.1 mg/kg) induced a robust neuronal silencing effect exclusively in hM4D Tg mice. These findings clearly demonstrate that CLZ, not CNO, is the primary contributor to in vivo imaging signals and pharmacological effects in murine DREADD models. Additionally, our study confirms that the original hM4D Tg mouse line is a suitable model with stable DREADD expression for developing novel DREADD agonists.
Keywords: Designer Receptors Exclusively Activated by Designer Drugs (DREADD), Human M4 mAChR DREADD (hM4D), ex vivo autoradiography, Clozapine (CLZ), Clozapine-N-oxide (CNO)
Received: 22 Jul 2025; Accepted: 27 Aug 2025.
Copyright: © 2025 Wang, Qin, Kumata, Zhang, Qian, Zhou and JI. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Jun Qian, Fudan University, Shanghai, China
Wen Zhou, Tianjin Medical University, Tianjin, China
BIN JI, Fudan University, Shanghai, China
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