
==== Front
Acta Pharm Sin B
Acta Pharm Sin B
Acta Pharmaceutica Sinica. B
2211-3835
2211-3843
Elsevier

S2211-3835(24)00229-6
10.1016/j.apsb.2024.05.033
Original Article
Discovery and evaluation of a novel 18F-labeled vasopressin 1a receptor PET ligand with peripheral binding specificity
Hu Junqi a†
Li Yinlong b†
Dong Chenchen a
Wei Huiyi a
Liao Kai a
Wei Junjie a
Zhao Chunyu b
Chaudhary Ahmad b
Chen Jiahui b
Xu Hao a
Zhong Ke c
Liang Steven H. steven.liang@emory.edu
b⁎
Wang Lu l_wang1009@foxmail.com
l_wang1009@jnu.edu.cn
a⁎
Ye Weijian ywjky@foxmail.com
a⁎
a Center of Cyclotron and PET Radiopharmaceuticals, Department of Nuclear Medicine & Key Laboratory of Basic and Translational Research on Radiopharmaceuticals, the First Affiliated Hospital of Jinan University, Guangzhou 510630, China
b Department of Radiology and Imaging Sciences, Emory University, Atlanta, GA 30322, USA
c Department of Pharmacy, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China
⁎ Corresponding authors. steven.liang@emory.edul_wang1009@foxmail.coml_wang1009@jnu.edu.cnywjky@foxmail.com
† These authors made equal contributions to this work.

03 6 2024
9 2024
03 6 2024
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19 1 2024
6 5 2024
13 5 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The arginine-vasopressin (AVP) hormone plays a pivotal role in regulating various physiological processes, such as hormone secretion, cardiovascular modulation, and social behavior. Recent studies have highlighted the V1a receptor as a promising therapeutic target. In-depth insights into V1a receptor-related pathologies, attained through in vivo imaging and quantification in both peripheral organs and the central nervous system (CNS), could significantly advance the development of effective V1a inhibitors. To address this need, we develop a novel V1a-targeted positron emission tomography (PET) ligand, [18F]V1A-2303 ([18F]8), which demonstrates favorable in vitro binding affinity and selectivity for the V1a receptor. Specific tracer binding in peripheral tissues was also confirmed through rigorous cell uptake studies, autoradiography, biodistribution assessments. Furthermore, [18F]8 was employed in PET imaging and arterial blood sampling studies in healthy rhesus monkeys to assess its brain permeability and specificity, whole-body distribution, and kinetic properties. Our research indicated [18F]8 as a valuable tool for noninvasively studying V1a receptors in peripheral organs, and as a foundational element for the development of next-generation, brain-penetrant ligands specifically designed for the CNS.

Graphical abstract

This work described the synthesis and evaluation of a novel 18F-labeled vasopressin 1a receptor PET ligand, which showed peripheral binding specificity and could cross the blood‒brain barrier in non-human primates.Image 1

Key words

Vasopressin
Autism
V1a receptor
Positron emission tomography
PET imaging
Radiolabeling
Fluorine 18
Kinetic modeling
==== Body
pmc1 Introduction

Arginine vasopressin (AVP), a unique cyclic nonapeptide hormone, plays a vital role in various physiological functions essential for maintaining homeostasis in both the periphery and the central nervous system (CNS)1,2. The physiological effects of AVP are predominantly mediated by three G-protein-coupled receptors: V1a, V1b, and V2, each exhibiting distinct distribution patterns and physiological impacts. The V1a receptor, predominantly found in vascular smooth muscle, liver, adrenal gland, brain, is associated with a range of peripheral and central pathologies3. Studies suggest that the V1a receptor influences not only metabolic regulation, blood pressure, and vascular resistance4, 5, 6, 7, 8, 9, 10, but also has garnered considerable attention regarding neurological conditions, social cognition and behavior11, 12, 13, 14. During the 1990s, studies revealed that social and anxiety-like behaviors in rats were found to be affected by intracerebroventricular and intraseptal injections of V1a-specific antagonists15, 16, 17, as well as by the administration of V1a antisense oligodeoxynucleotide into the septal region18, suggesting an association between V1a receptor and these behaviors. Subsequent research has further explored the role of V1a in social cognition and behavior19, 20, 21, and its potential implications for autism spectrum disorder (ASD)22, 23, 24, 25, 26, a complex neurodevelopmental condition characterized by impaired social interaction, communication challenges, and repetitive behaviors. Despite ongoing investigations into the detailed mechanisms, the V1a receptor has emerged as a potential therapeutic target in ASD27, 28, 29, 30, 31, 32.

The multifunctional nature of V1a receptor has driven extensive research, leading to the development of numerous antagonists33,34 (Fig. 1A). In the early 1990s, OPC-2126835 (1) emerged as the first V1a-selective antagonist, exhibiting high affinity for rat V1a receptor but weak affinity for human's SR 4905936 (2) was noted for its high affinity, selectivity and efficacy towards both animal and human V1a receptors. In the last decade, Azevan Pharmaceuticals developed SRX24637 (3), a highly selective and specific V1a receptor antagonist that proven to be safe and well-tolerated in a randomized Phase II clinical trial38. It can decrease anxiety-potentiated startle in healthy individuals39, and reduce aggressive behavior in irritable Huntington's disease patients40. Additionally, RG771341 and balovaptan42 (RG7314, 4), discovered by Roche, have progressed into advanced clinical studies involving individuals with autism. However, despite receiving Breakthrough Therapy Designation from FDA in 2018 and showing significant efficacy on the Vineland-II scale in ASD in a Phase II trial43, balovaptan did not demonstrate efficacy in enhancing socialization and communication in pediatric ASD patients44 or autistic adults45 in the subsequent Phase II or III trials. These outcomes underscore the complexity of the V1a signaling pathway and emphasize the necessity for further mechanistic studies. Hence, in vivo investigation of the V1a receptor system and its interaction with potential drug candidates via non-invasive techniques becomes imperative, offering invaluable biology insights and contributing to a comprehensive understanding of their roles and therapeutic potential.Figure 1 Representative antagonists (A) and radiolabeled ligands (B) for V1a receptor.

Figure 1

Positron emission tomography (PET), featuring the capacity for in vivo imaging and quantifying biochemical processes in real-time, emerges as a promising solution for studying intricate physiological and pathological mechanisms without the need for invasive operations. The development of novel PET ligands targeting the V1a receptor remains pivotal in unraveling the complexities of physiological and pathological processes, especially in the regulation of social, emotional, cognitive behaviors. Recent advancement has been made in developing radiolabeled V1a ligands (Fig. 1B), including analogs of SRX24646 ([123I]5, [18F]5 and [11C]5), [11C]647 and our work [11C]PF-18456348 ([11C]7). While [11C]PF-184563 stands out as the first PET ligand suitable for V1a receptor imaging in peripheral organs, there remains a need for ideal PET ligands, especially for applications both in the CNS and periphery.

This study aims to develop and validate an effective V1a-targeted PET ligand with enhanced blood‒brain barrier (BBB) penetration through structural optimization. Encouraged by the results of [11C]PF-184563, we hypothesized that, by maintaining binding affinity, the integration of 18F into the pyridine scaffold could not only prolong the ligand half-life from 20.4 to 109.8 min but also enhance BBB permeability due to inductive effect and the shielding of the nitrogen atom on the pyridine ring. In this report, we describe the design, synthesis, pharmacological profiling, radiolabeling, and comprehensive in vitro and in vivo characterization of a novel radiolabeled analog, [18F]V1A-2303 ([18F]8), as a potential probe for PET imaging of the V1a receptor.

2 Result and discussion

2.1 Chemical synthesis

The target compound 8 was synthesized in a similar fashion based on our previous report48. As outlined in Scheme 1, the reaction commenced with commercially available compounds methyl piperidine-4-carboxylate 9 and 2,6-difluoropyridine 10. Nucleophilic aromatic substitution reactions were executed to generate 11. Subsequent steps involving hydrolysis, esterification, and hydrazinolysis, resulted in hydrazine 13 in good yield. The intermolecular cyclization of hydrazine 13 with thioamide 14 afforded 1,2,4-triazole analogue 15 in 26% yield. Boc group deprotection under acidic conditions, followed by reductive amination, led to the desired compounds 8 in 52% yield over two steps. The new compounds were fully characterized by 1H and 13C NMR spectroscopy and MS.Scheme 1 Synthesis of target compound 8.

Scheme 1

2.2 Binding affinity and BBB permeability

Having obtained target compound 8, its biological activity and pharmacological properties were assessed through in vitro assays. As outlined in Table 1, this compound exhibited nanomolar potency and high affinity for the hV1a receptor (IC50 = 2.33 nmol/L and Ki = 0.46 nmol/L), which were determined using 0.03 nmol/L [125I] phenylacetyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 as radioligand, with specific binding at 85% and a Bmax value of 2.80 pmol/mg protein. Furthermore, its binding affinities for other vasopressin receptors (V1b and V2) exceeded 10 μmol/L, highlighting excellent target selectivity.Table 1 Pharmacological and physiochemical properties of target compound 8.

Table 1Pharmacology properties	Value	Physiochemical properties	Value	
hV1A IC50(nmol/L)	2.33	Mw (g/mol)	412.9	
hV1A Ki(nmol/L)	0.46	LogD (pH 7.4)	1.81 ± 0.01	
hV1B IC50(nmol/L)	>10,000	Papp [A‒B]( × 10−6 cm/s)	33.48	
hV2 IC50(nmol/L)	>10,000	Efflux ratio	0.85	
		LogBB	‒0.09	
		MPO score	5.3	

To predict BBB permeability, the lipophilicity of compound 8 was evaluated using the shake-flask method, yielding a logD7.4 of 1.81 ± 0.01 (n = 3). This suggests that it may be suitable as a ligand for PET imaging in the brain since molecules typically pass through the BBB via passive diffusion when logP or logD values fall between 1 and 3.549. MDCK-MDR1 experiments showed an apparent permeability coefficient Papp[A‒B] of 33.48 × 10−6 cm/s (values > 10 × 10−6 cm/s suggesting high BBB permeability), and an MDR1 efflux ratio (ER) of 0.85, suggesting that the compound 8 likely possesses the ability to penetrate the brain. Additionally, logBB is a standardized measurement of BBB permeability (the logarithmic ratio between the concentration of a compound in the brain and blood), and the logBB values between 0.3 and −1 represent the compound that could pass the BBB50,51. The logBB value of compound 8 was predicted as −0.09, which suggests compound 8 possesses the ability to penetrate BBB. Likewise, multiparameter optimization (MPO) is a desirability tool to assess the BBB-permeability of drug candidates. Generally, a CNS MPO score on a scale between 0 and 6, and a score value ≥ 4.0 is highly desirable52. The calculated MPO score for compound 8 was 5.3, indicating compound 8 as a potential candidate. Moreover, ex vivo measurements53 of BBB permeability in rat brain tissues and plasma by LC‒MS/MS further supported this, demonstrating compound 8 can traverse the BBB (Supporting Information Table S1).

2.3 Radiochemistry

The radiolabeled compound, [18F]8, was designed to be synthesized through SNAr reactions54 of the corresponding precursor featuring a nitro leaving group, 22. Following a synthesis route similar to that of compound 8 but incorporating 2-fluoro-6-nitropyridine (17), the reaction steps included substitution, hydrazinolysis, intermolecular cyclization, deprotection, and reductive amination, yielding the compound 22 in 2.5% yield over five steps (Scheme 2). Upon heating with [18F]TEAF/TEAB at 180 °C for 30 min, the labeling precursor 22 smoothly transformed into [18F]8. Employing an automated synthesis module encompassing radiolabeling, purification, and formulation, [18F]8 was obtained with a non-decay-corrected radiochemical yield of 7.9 ± 2.2% (n ≥ 3) within 100 min. The product exhibited >95% radiochemical purity and a molar activity >58.8 GBq/μmol (Supporting Information Fig. S1) at the end of synthesis (EOS). The in vitro stability of the radiotracer was investigated by incubation at 37 °C in saline containing 10% EtOH (Supporting Information Fig. S2A) or monkey plasma samples (Fig. S2B). As expected, [18F]8 was proved to be stable in these media, with no degradation observed by radio-HPLC up to 120 min of incubation time. The ultrafiltration method showed an unbound free fraction (fu) of [18F]V1A-2303 as 34.4 ± 2.2% using non-human primate (NHP) plasma.Scheme 2 Synthesis of radiolabeled V1a ligand [18F]V1A-2303.

Scheme 2

2.4 Cell uptake assay, ex vivo and in vitro autoradiography

To assess the in vitro specificity of [18F]8 to the V1a receptor, cell uptake studies were conducted. Chinese hamster ovary (CHO) cells expressing the human V1a receptor were incubated with [18F]8 alone or in combination with a more than 100-fold excess amount of the non-radioactive compound 8 and V1a antagonists PF-184563, Balovaptan and SRX246 (Fig. 2A). Both structurally similar V1a antagonists (PF-184563, Balovaptan) and structurally different V1a antagonists (SRX246) resulted in a substantial reduction in [18F]8 cell uptake, indicating the specific binding of [18F]8 to the V1a receptor in vitro.Figure 2 In vitro cell uptake assay and autoradiography of [18F]8. (A) Normalized uptake ratio of [18F]V1A-2303 in CHO cell line that expresses the human V1a receptor, in the presence of non-radioactive V1A-2303, V1a receptor antagonists PF-184563, balovaptan or SRX246 as blocking agents (0.5 μmol/L); Representative images for baseline and blocking (10 μmol/L) by (B) ex vivo autoradiography in mice liver and (D) in vitro autoradiography in monkey adrenal; (C, E) Comparative analysis under baseline and blocking conditions to quantitatively assess the specificity of [18F]V1A-2303. All data are shown as mean ± SD (n ≥ 3) and analyzed by unpaired t-test. ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Figure 2

To further explore the binding specificity of [18F]8 at tissue levels, ex vivo autoradiography studies were performed on liver slices from C57BL/6 mice, and in vitro autoradiography studies were conducted on brain sections from rats and adrenal gland sections from monkeys. Under baseline conditions, high signal intensities were observed in liver sections (Fig. 2B). Pretreatment with balovaptan (10 mg/kg, i.v.) led to a roughly 50% reduction in signal intensities (Fig. 2C). For brain slices, while relative signal intensity only decreased by 8.5%, there was a statistically significant difference between the baseline and blocking conditions (Supporting Information Fig. S3). Because the V1a receptor density in the brain is low55, 56, 57, 58 may not be sufficiently high to accurately reflect specific V1a receptor binding in the brain. Furthermore, adrenal gland sections from monkeys incubated with [18F]8 exhibited high specific binding in the adrenal cortex (zona glomerulosa and zona fasciculata) and medulla (Fig. 2D), regions known to express the V1a receptor59. The signal intensities significantly decreased with the blockade of V1a antagonists V1A-2303 or PF-184563 (10 μmol/L; Fig. 2E). Compared to the blocking studies using unlabeled V1A-2303, the substantial activity retention observed in rhesus monkey adrenals under PF-184563 blocking conditions could be attributed to the residual nonspecific binding. These findings further demonstrate that [18F]8 binds specifically to the V1a receptor, and this binding is effectively blocked by various V1a antagonists.

2.5 Biodistribution study in mice

To study the pharmacokinetic profile of [18F]8, we assessed its uptake, distribution, and elimination in C57BL/6 mice at five-time points (1, 5, 15, 30, and 60 min) post-injection (p.i.). The results are presented in Fig. 3A and Supporting Information Table S2 as the percentage of the injected dose per gram of wet tissue (%ID/g). The initial clearance of radioactivity from the blood was rapid, exhibiting a clearance ratio of 2.3 between 1 and 60 min. Several organs, including the heart, lung, liver, pancreas, small intestine, and kidney, displayed high radioactivity levels (>5%ID/g) at initial time points. Subsequently, radioactivity rapidly decreased in most tissues, except for the liver, where it initially increased until the 5 min p.i. before gradually diminishing. The radioactivity in the stomach and small intestine plateaued at 30 min p.i., while significant radioactivity persisted in the bladder at 60 min p.i. The relatively elevated uptake in the liver and small intestine may be attributed to hepatobiliary excretion, along with potential intestinal reabsorption. Meanwhile, the relatively constant uptake in the kidneys and gradual accumulation in the bladder suggests renal clearance.Figure 3 Ex vivo biodistribution of [18F]8 in mice. (A) Biodistribution of tracer [18F]8 at five different time points characterized by injected dose per gram of wet tissue (%ID/g) under baseline condition. The inset shows the brain uptake; (B) Standard uptake value (SUV) in the brain at five different time points; and comparison of tracer uptake in the liver (C) and kidney (D) under baseline and blocking conditions. V1a antagonist balovaptan (10 mg/kg) was used as a blocking agent. All data were shown as mean ± standard deviations (n = 4) and analyzed by paired t-test. nsP＞0.05, ∗P ≤ 0.05.

Figure 3

The brain uptake was low at 0.91 %ID/g (SUV = 0.18) at 1 min p.i. and gradually diminishing over 60 min (Fig. 3A and B), likely influenced by P-glycoprotein (P-gp) efflux in mouse brain (Supporting Information Fig. S4). An ex vivo brain metabolism study, analyzing radio chromatograms at 5 and 30 min p.i., showed good stability and a modest rate of metabolization of [18F]8 in the brain (Supporting Information Fig. S5, Tables S3 and S4). The intact parent fractions in the brain were 90.3 ± 1.2% at 5 min and 78.8 ± 0.4% at 30 min, indicating that the majority of brain uptake likely originated from the probe itself rather than its metabolites. However, the ex vivo analysis of liver metabolism revealed that, at 30 min, 26.9 ± 1.69% of the signal in the liver originated from the parent radiotracer, while the remaining 73% corresponded to radio metabolites (Supporting Information Fig. S6). We noted a progressive buildup of radioactivity in bone, suggesting potential defluorination of the tracer in rodents.

When pre-treating with balovaptan (10 mg/kg) before the injection of radiolabeled tracer [18F]8, the radioactivity in most organs and tissues decreased at 5 min and 30 min p.i (Supporting Information Fig. S7 and Table S5). The radioactivity in the liver declined by 60% at 5 min and 54% at 30 min (Fig. 3C), while the radioactivity in the kidney declined by 30% at 5 min and 10% at 30 min (Fig. 3D), in the comparison with baseline study. However, the changes without statistical significance in the kidney indicate that the uptake is mainly attributed to excretion rather than binding, while the significant change in the liver may exhibit high binding specificity.

2.6 PET imaging studies of [18F]V1A-2303 in monkey

Dynamic PET imaging studies were conducted in the rhesus monkey's brain to assess brain permeability, binding specificity, and washout kinetics of [18F]8. Fig. 4A illustrates the co-registration of averaged PET images of the brain (0–90 min) with the MRI T1-weighted template, and Fig. 4B displays the time-activity curves (TACs) of specific brain regions. The baseline study indicated that [18F]8 was able to cross the blood‒brain barrier, with a peak whole-brain standard uptake value (SUV) of 1.6 at 2.5 min. Radioactivity in all regions of interest exhibited a rapid increase, peaked at 2.5 min, and was gradually washed out over 100 min. Specifically, there was a heterogeneous distribution of radioactivity, with the highest level in the pituitary gland, followed by the thalamus, striatum, amygdala, hippocampus, and cerebellum with the least. In Fig. 4C, static whole-body maximum intensity projection (MIP) images acquired 100–110 min p.i. are presented. There was some radioactivity in the vertebrae and potential joints, suggesting potential defluorination of the tracer in non-human primates (NHPs). Comparing results with and without pretreatment of balovaptan (3 mg/kg), a notable accumulation was observed in the liver in the baseline, with some activity extending to the intestine, suggesting a preference for hepatobiliary excretion over renal clearance, which was similar to the biodistribution results in rodents. Following pretreatment with balovaptan, a notable reduction in the liver signal was observed, with the SUV in the liver decreasing from 11 to 5 (Fig. 4D and E), indicating a pronounced blockade effect on the hepatic uptake. However, pretreatment with V1a antagonist balovaptan (3 mg/kg) did not decrease the signal in various brain regions, yet resulted in increased radioactivity and a higher whole-brain area under the curve (AUC) (Supporting Information Fig. S8 and Table S6).Figure 4 PET imaging studies of [18F]8 in the rhesus monkey. (A) Representative summed PET/MR images (0–90 min) of the brain under baseline conditions; (B) Time-activity curves (TACs) in representative brain regions under baseline conditions; (C) Whole body maximum intensity projection (MIP) images; (D) PET/CT static images (100–110 min) of liver; (E) Liver mean standard uptake value (SUVmean) of monkey after 100 min injection under both baseline and blocking conditions. Blocking was pre-treated with balovaptan (3 mg/kg).

Figure 4

2.7 Kinetic modeling in NHP brain

We employed compartmental modeling with a metabolite-corrected input function to determine brain kinetic parameters. Following intravenous bolus injection of [18F]8, plasma radioactivity exhibited a transient peak within 1 min, followed by a substantial decrease (Fig. 5A). The pre-treatment of balovaptan (3 mg/kg) resulted in a slight increase in peak activity and AUC. The plasma-to-whole blood ratio remained relatively stable, averaging around 1.01 ± 0.05 (Supporting Information Fig. S9 and Table S7). [18F]8 exhibited reasonable metabolism in arterial blood, with intact parent fractions of 66.1% at 10 min and 38.6% at 30 min (Fig. 5B). Balovaptan pre-treatment slightly elevated the intact parent fraction (74.4% at 10 min and 47.5% at 30 min), indicating increased plasma availability of the tracer induced by balovaptan. This observation aligns with the hypothesis that balovaptan exerts a more extensive blockade effect on peripheral receptors than in the brain. As illustrated in Fig. 5C and Supporting Information Table S8, radio-chromatogram analyses revealed that [18F]8 had a retention time of 10 min. The primary polar radioactive metabolite exhibited a shorter retention time, implying a reduced likelihood of penetrating the BBB compared to the parent tracer.Figure 5 Plasma data, kinetic modeling, and time stability analysis in the brains of non-human primates (NHPs). (A) Individual metabolite-corrected [18F]V1A-2303 standard uptake value (SUV) time courses in plasma; (B) Individual fraction of unchanged parent compound over time; (C) Representative radio-chromatograms of plasma samples under baseline conditions; (D, E) Model fits of one-tissue compartment model (1TCM), two-tissue compartment model (2TCM), and Logan plot on the baseline scan of monkey striatum. (F) Estimation of total volume of distribution (VT) calculated for 60–100 min scan windows in 10 min increments compared to the final 90 min VT for the thalamus and striatum.

Figure 5

The representative kinetic modeling results, along with detailed parameters obtained from various fitting models for all volumes of interest (VOIs), are summarized in Table 2. The two-compartment model (2TCM) consistently outperformed the one-compartment model (1TCM) for all evaluated VOIs, evident from the lower Akaike Information Criterion (AIC) (Fig. 5D). Under baseline conditions, the amygdala exhibited the highest total volume of distribution (VT) of 1.26, followed by the thalamus, striatum, and hippocampus, with the cerebellum displaying the lowest VT. Under blocking condition, administration of V1a antagonist balovaptan (3 mg/kg) before tracer injection enhanced the VT values of analyzed brain regions and indicated an absence of specific binding in the brain. Fig. 5E illustrates the Logan plot for the striatum, highlighting a robust correlation between VT values derived from 2TCM and Logan plots (Pearson's r = 0.941, P < 0.0001, Supporting Information Fig. S10). Furthermore, Supporting Information Table S9 outlines the VT values for each brain region across five different time intervals, ranging from 0 to 60 to 0–100 min p.i. The stability of VT in the thalamus and striatum is depicted in Fig. 5F. During the 60–100 min interval, VT values calculated using 2TCM fitting exhibited minimal fluctuation, remaining between 90% and 100%. This consistency suggests that VT stabilizes after 60 min in the dynamic PET imaging scans. These results suggest that 2TCM constitutes a suitable model for the assessment of [18F]8 kinetic parameters in the NHP brain.Table 2 Pharmacokinetic parameters estimated from different fitting models.

Table 2Region	K1 (mL/cm3/min)	k2 (/min)	k3 (/min)	k4 (/min)	VT (mL/cm3)	VT (block)a	
2TCM	
Thalamus	0.10 ± 0.03	0.15 ± 0.03	0.05 ± 0.01	0.09 ± 0.05	1.14 ± 0.35	1.95	
Striatum	0.09 ± 0.03	0.15 ± 0.03	0.04 ± 0.02	0.05 ± 0.002	1.11 ± 0.32	1.86	
Occipital cortex	0.10 ± 0.02	0.21 ± 0.01	0.03 ± 0.0002	0.02 ± 0.01	1.10 ± 0.47	1.53	
Amygdala	0.09 ± 0.04	0.15 ± 0.05	0.03 ± 0.02	0.02 ± 0.002	1.26 ± 0.42	1.73	
Hippocampus	0.09 ± 0.04	0.16 ± 0.06	0.03 ± 0.02	0.03 ± 0.01	1.13 ± 0.31	1.68	
Cerebellum	0.10 ± 0.03	0.23 ± 0.03	0.04 ± 0.001	0.03 ± 0.01	0.97 ± 0.39	1.48	
1TCM	
Thalamus	0.09 ± 0.02	0.09 ± 0.004			0.99 ± 0.21	1.93	
Striatum	0.08 ± 0.02	0.09 ± 0.005			0.93 ± 0.22	1.78	
Occipital cortex	0.09 ± 0.01	0.14 ± 0.01			0.65 ± 0.71	1.34	
Amygdala	0.07 ± 0.03	0.08 ± 0.01			0.87 ± 0.23	1.62	
Hippocampus	0.08 ± 0.03	0.09 ± 0.01			0.82 ± 0.19	1.60	
Cerebellum	0.08 ± 0.02	0.13 ± 0.007			0.61 ± 0.18	1.36	
Logan plot	
Thalamus					1.18 ± 0.36	1.96	
Striatum					1.14 ± 0.34	1.88	
Occipital cortex					1.10 ± 0.43	1.54	
Amygdala					1.33 ± 0.55	1.69	
Hippocampus					1.16 ± 0.38	1.71	
Cerebellum					1.03 ± 0.41	1.53	
Data are presented as mean ± SD.

a Pre-blocking studies were performed with 3 mg/kg of balovaptan.

V1a receptor plays a critical role in the regulation of a range of physiological processes, and its dysregulation influences the pathophysiology of various neurological conditions, social cognition and behavior. Moreover, the impact of the V1a receptor extends beyond the CNS to numerous peripheral pathologies, including metabolic regulation, blood pressure, and vascular resistance. Recent studies have also considered the V1a receptor as a potential therapeutic target for castration-resistant prostate cancer. The wide-ranging physiological impacts of the V1a receptor highlight its potential as a therapeutic target for both central and peripheral diseases.

V1A-2303 was designed and synthesized as a V1a receptor-targeted ligand. In vitro pharmacological characterization revealed an IC50 of 2.33 nmol/L and a Ki of 0.46 nmol/L for hV1a, a logD7.4 of 1.81, and an ER of 0.85. Balovaptan42, for comparison, exhibited a Kb of 0.23 nmol/L for hV1a, a logD7.4 of 3.2, and a hP-gp efflux ratio of 2.3. Additionally, the binding affinity of V1A-2303 is higher than the other V1a-targeted radio ligands reported in the literature. Based on these pharmacological parameters, V1A-2303 can be considered a V1a receptor-targeted ligand similar to balovaptan.

In the biodistribution study in mice, a low brain uptake (0.91 %ID/g, SUV = 0.18 at 1 min p.i.) indicated poor brain permeability. However, moderate brain uptake was observed in PET imaging of rhesus monkeys (SUV = 1.6 at 2.5 min p.i.), approximately 8-fold higher than in mice. This significant difference is likely attributed to species variations in efflux transporter expression in the brain, as the expression of the efflux transporter MDR1 was 2.33-fold lower in humans than in mice60. While mice studies are an important step in the development of CNS-targeted tracers, it is crucial to remember that they do not always accurately predict efficacy in higher species. Further research in NHP models and even humans is necessary to fully evaluate their properties.

[18F]8 exhibits good stability and reasonable metabolization rates. In the ex vivo brain metabolism study, intact parent fractions of [18F]8 were 90.3 ± 1.2% at 5 min and 78.8 ± 0.4% at 30 min. Pharmacokinetic results, brain tissue distribution study, and radio-chromatogram analyses supported that the majority of brain uptake in biodistribution and PET imaging studies likely originated from the probe itself rather than its metabolites. In kinetic modeling, intact parent fractions of [18F]8 in plasma were 66.1% at 10 min and 38.6% at 30 min, comparable to [18F]LW22361 (a TSPO-targeted PET radioligand, ∼50% at 10 min and ∼40% at 30 min) and superior to 18F-AV4562 (the first FDA-approved commercial radiotracer for Aβ PET imaging, 35 ± 10% at 10 min and 14.4 ± 7.3% at 30 min). Consequently, blood data from monkey experiments suggests modest metabolization of [18F]8.

In the PET imaging studies of [18F]8 in monkeys, pretreatment with V1a antagonist balovaptan notably decreased the signal in the liver, but did not decrease the signal in various brain regions. The SUV in the pituitary (Fig. 4B and Supporting Information Fig. S8) showed a slight increase at both 10 min (from 1.25 to 1.30) and 30 min (from 0.91 to 1.03) compared to the non-pretreated group. The AUC (0–90 min) value of the time-activity curve of metabolite-corrected pituitary increased by 5.9%. The increase caused by the pretreatment with balovaptan can be attributed to three factors: 1) The increased radioactivity in plasma. On one hand, the peak SUV in the metabolite-corrected plasma (Fig. 5A) is 37.11 at 0.267 min in baseline and 44.52 at 0.517 min (an 18.9% increase) in blocking. On the other hand, the area (Supporting Information Table S10) under the metabolite-corrected plasma time‒activity curve (0–90 min) increased by 20% (from 57.04 to 68.40); 2) The increased metabolic stability of [18F]8. As shown in Fig. 5B, the pre-treatment of balovaptan increased the intact parent fraction in plasma by 12.6% at 10 min and 23.2% at 30 min; 3) The increased unbound free fraction (fu or fp). This factor is directly related to the ability of the radioligand to cross the blood-brain barrier and bind to its target. It was reported that the mean fp value of blocked scans was likely higher than that of baseline scans63. As previously discussed, the administration of the blocking agent altered the intact parent fraction in plasma, which showed higher concentrations of [18F]8 compared to baseline, likely due to the displacement of the radioligand from peripheral binding sites. This suggests that fu or fp may be significantly increased in the presence of pharmacological dose of blocker.

PET is a promising tool for studying complex physiological and pathological mechanisms, and the development of innovative PET tracers holds the potential to improve diagnostics, personalize treatment approaches, and advance our comprehension of various diseases. A prime example is the development of TSPO radioligands. The latest radioligands are insensitive to the rs6971 polymorphism, which significantly alters the affinity of TSPO for drug and probe, and enables the visualization of in vivo preclinical brain inflammation. This example sets the stage for further investigations of the V1a receptor. Similarly, the single nucleotide polymorphisms (rs11174811) and microsatellite polymorphisms (RS1 and RS3)26,64, 65, 66 of V1a, associated with autism, pair bonding-behavior, maternal behavior, and other aspects of social cognition and behavior, may shed light on individual differences in social behavior and heritable disorders. Hence, the ongoing development of novel PET ligands targeting the V1a receptor is crucial for unraveling the complexities of physiological and pathological processes, especially in regulating of social, emotional, and cognitive behaviors. Such progress will facilitate the translation of findings from animal studies on prosocial behavior to preclinical human research.

3 Conclusions

We synthesized [18F]V1A-2303 ([18F]8), a PET ligand for the V1a receptor. [18F]8 exhibited favorable pharmacological characteristics, including high binding affinity (Ki = 0.46 nmol/L), excellent selectivity (>100-fold for V1a over other AVP receptors), and efficient BBB permeability. Autoradiography and biodistribution studies demonstrated good specificity for the V1a receptor in peripheral tissues. [18F]8 also showed high specific binding in the liver and was able to cross the BBB in NHPs as determined by PET imaging. However, kinetic modeling using a metabolite-corrected arterial input function revealed a lack of specific binding in the brain. Further medicinal chemistry efforts are underway and we aim to increase binding affinity in 10‒30-fold based on our brain-penetrant ligand scaffold and test if moderate specific binding could be achieved under these conditions to enable V1a imaging in the CNS.

4 Experimental

4.1 Reagents

Unless otherwise indicated, all reagents were acquired from Sigma‒Aldrich and solvents were newly dried and degassed before use.

4.2 Animals

Sprague‒Dawley (SD) rats (male, 7–8 weeks, 220–240 g) were purchased from Charles River Laboratory, and C57BL/6 mice (male, 7–8 weeks, 20–25 g) were purchased from GuangDong GemPharmatech Co., Ltd. All animals received humane care and were housed in cages with a 12 h light/dark schedule. In compliance with Jinan University's Animal Ethics Committee, the rodent experiment protocols have been approved. Several studies on rhesus monkeys (male, six years old, 6–7 kg) were approved by the Institutional Animal Care and Use Committee (IACUC) of Guangdong Landau Biotechnology Co., Ltd. This organization is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

4.3 Chemical synthesis

Details of synthesis procedures and NMR data are included in the Supporting Information.

4.4 Pharmacology and ADME properties

4.4.1 Binding affinity and selectivity assays for V1A-2303 (8)

All pharmacology studies were performed at Eurofins BioPharma Services. In vitro inhibitory effects of compound V1A-2303 on V1a, V1b, and V2 receptors were determined using established commercial assays. Reference standards were included in every assay to verify the accuracy of the results. The determination of IC50 values was conducted through a non-linear, least squares regression analysis employing MathIQTM (ID Business Solutions Ltd., UK). In cases where inhibition constants (Ki) were reported, the Ki values were calculated using the Cheng and Prusoff equation67. The presentation of IC50 and Ki values does not include the Standard Error of the Mean (SEM).

General experimental procedure (V1A assays as an example): Human recombinant vasopressin V1A receptors expressed in HEK-293 cells are used in modified Tris-HCl buffer pH 7.4. A 0.26 μg aliquot is incubated with 0.03 nmol/L [125I]phenylacetyl-d-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 for 120 min at 25 °C. Non-specific binding is estimated in the presence of 1 μmol/L [Arg8]vasopressin. Receptors are filtered and washed, the filters are then counted to determine [125I]phenylacetyl-d-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 specifically bound.

4.4.2 Measurement of lipophilicity

[18F]V1A-2303 was formulated into a 15 mL tube containing n-octanol (3.65 mL, 3 g) and PBS (3 mL, 3 g) that were pre-saturated. When the vortex has been vortexing for 3 min, for better liquid layering, the hybrid was divided into three new tubes and separated by centrifugation for 5 min (3500 rpm). In the following steps, every aliquot's count per min (CPM) was determined using an automatic gamma counter (WIZARD2 2480, PerkinElmer, USA), and decay and background corrections were made. To calculate logD, we divided the radioactivity between the organic phase and the aqueous phase.

4.4.3 MDCK-MDR1 experiment

In vitro brain permeability of V1A-2303 was carried out using a Madin Darby canine kidney (MDCK) cells with the MDR1 gene model by methods68. Reference compounds are digoxin (a P-glycoprotein drug efflux transporter control), metoprolol (a high permeable control), atenolol (a low permeable control), and Lucifer Yellow. Hank's balanced salt solution (HBSS) buffer along with 5 mmol/L Lucifer Yellow and 0.4% DMSO (pH 7.4) was used to wash MDR1-MDCK cells twice before transport experiments.

4.4.4 Ex vivo permeability of BBB in SD rats

These studies were performed at ChemPartner. SD rats (220–240 g, male, n = 9) were administered intravenously at a dose of V1A-2303 (0.03 mg/kg) via foot dorsal vein injection. At the time points of 5, 30, and 60 min, a blood sample of nearly 150 μL was collected from the animal. Immediately following collection, the blood samples were kept on ice and centrifuged (2000×g, 4 °C, 5 min) to obtain plasma. As soon as the animals had been anesthetized and exsanguinated, tissue samples (pituitary, brainstem, hippocampus, striatum, and cortex) were collected, rinsed with pre-cooled saline, dried on filtrate paper, weighed, and frozen on dry ice. Tissue samples were homogenized with 3 volumes (v/w) of homogenizing solution (PBS) before analysis. The concentrations of V1A-2303 in samples were quantified through liquid chromatography-tandem mass spectrometry (LC‒MS/MS-39, Triple Quad 6500+). The detection of all analytes was carried out in positive ionization mode. The MRM (multiple reaction monitoring) transitions were m/z 413.10 → 177.20 for V1A-2303 and m/z 230.00 → 121.00 for Osalmid (internal standard).

4.5 Radiochemistry

[18F]Fluoride was generated via the 18O(p,n)18F reaction in a 10 MeV cyclotron (GE, Qilin) using >98% enriched H218O (Taiyo Nippon Sanso Corporation, Tokyo, Japan). [18F]Fluoride was trapped with a Sep-Pak QMA cartridge (Waters, 186004540), and eluted with TEAHCO3 (2.0 mg) in MeOH (1 mL). After an azeotropic drying, precursor 22 (0.5 mg) in anhydrous DMSO (500 μL) was added. The radiosynthesis occurred at 180 °C for 30 min, and was followed by HPLC purification with an Osaka Soda CAPCELL PAK C18 column (UG80, 5 μm, 10 mm i.d. × 250 mm) using a mobile phase of CH3CN/H2O (v/v, 45/55) containing 0.1% Et3N and at 3.0 mL/min. The retention time (tR) of [18F]8 was 18.0 min. The product fraction was collected, diluted with water, and trapped with a C-18 light cartridge (Waters, WAT023561). Eluted with 1 mL EtOH and diluted with 9 mL saline, the final product was obtained. Analytical HPLC, with a GL Sciences WondaSil C18-WR column (4.6 mm × 150 mm) and the same mobile phase at a flow rate of 1 mL/min (tR: 6.7 min), confirmed the identity of [18F]8 by co-injection with the reference compound 8.

4.6 Cell uptake studies

The CHO-V1a cell line, expressing the Human V1a Receptor, was cultured in Ham's F12 medium (Life Technologies) with 10% fetal bovine serum (Corning), 1% penicillin-streptomycin (Life Technologies) and Geneticin (G418, 0.4 mg/mL, Life Technologies). Maintained at 37 °C with 5% CO2 and high humidity, subpassaging involved a 1:3 split using 0.25% trypsin/0.02% EDTA. For cell uptake, CHO-V1a cells were plated at 2 × 105 cells/well for 24 h, exposed to [18F]V1A-2303, and incubated for 60 min. After PBS washes, cells were harvested with 0.1 mol/L NaOH. The blocking assay included a nonradioactive reference or PF-184563, Balovaptan, and SRX246 incubated for 60 min. Gamma counter measurements assessed cell uptake (%AD). Data represent averages from quadruplicate wells.

4.7 In vitro stability and fu in plasma

The in vitro stability of [18F]V1A-2303 was investigated by incubation at room temperature in saline containing 10% EtOH or monkey plasma for 0, 1 and, 2 h. Radioactivity in the mixture was detected by analytical HPLC.

The unbound fraction of [18F]V1A-2303 in NHP plasma samples was determined using an ultrafiltration method69. Arterial blood (2.5 mL) was drawn from the monkey, then centrifuged (5 min, 14,000 rpm, 4 °C) to obtain plasma. We added a solution of [18F]V1A-2303 (185 kBq) quadruplicately to the plasma (150 μL). Each tube was incubated for 10 min at 37 °C and then added with 300 μL cold PBS (pH 7.4) and vortexed for 15 s. We centrifuged the 460 μL mixture in a centrifuge tube containing an Amicon® Ultra-0.5 mL device (Merck Millipore Ltd. Tullagreen, Carrigtwohill, Co. Cork, IRELAND) for 20 min (14,000×g, 4 °C). To get the concentrated solution, the filter was removed and inverted into a new tube, and centrifuged again (1000×g for 2 min at 4 °C). Each section of the sample was measured using an automatic gamma counter (WIZARD2 2480, PerkinElmer, USA). The fu value was calculated as Eq. (1):(1) fu (%) = 1 ‒ (CPM of filter membrane) / [(CPM of filter solution) + (CPM of concentrated solution) + (CPM of filter membrane)] × 100

4.8 Autoradiography of ex vivo in mice liver and in vitro in monkey adrenal

[18F]V1A-2303 (7.4 MBq per animal) was administered intravenously via the tail vein to one group of C57BL/6 mice (male, 8 weeks, 25 g, n = 3). In another group, to determine the receptor-specific binding in the liver, mice were pretreated with balovaptan (10 mg/kg, i.v.) before the injection of tracer. Cervical dislocation was performed 30 min p.i. to sacrifice mice. After the liver parts were immediately removed, they were embedded in Tissue-Tek® (O.C.T.). Liquid nitrogen was used to freeze the tissue, and 20 μm sections were cut using a cryotome. A 12-h exposure to a phosphor imager plate followed.

Several sections of monkey adrenal tissue from cynomolgus macaques (male, n = 3) were stored at −80 °C until further processing. Briefly, sections were preconditioned with 200 mL buffer1 (50 mmol/L Tris containing 0.1% BSA) in a reservoir for 10 min. Afterward, incubation of the sections with 200 mL [18F]V1A-2303 solution (37 kBq/mL in buffer1) for 1 h was conducted. For baseline conditions, sections were dipped into the above solution and 10 μmol/L DMSO. For blockade, 10 μmol/L of V1a receptor antagonist, PF-184563, was added. In the following step, sections were transferred to buffer 2 (50 mmol/L Tris without 0.1%BSA) and washed. A 12-h exposure to a phosphor imager plate followed.

Imaging plates (BAS-MS2025, GE Healthcare, NJ, USA), an imager (Amersham Typhoon, Cytiva, USA), and a Bio-Imaging Analyzer System (Multi Gauge Version 2.3, FUJIFILM) were used to obtain autoradiograms and measure photostimulated luminescence (PSL) values in ROIs.

4.9 Biodistribution studies in mice

In this study, C57BL/6 mice (male, 7 weeks, 20–25 g) were used. Via the tail vein, each mouse received the solution of [18F]V1A-2303 (3.7 MBq/100 μL). At each time point (1, 5, 15, 30, and 60 min) p.i., four mice were dissected to get a whole brain, thymus, heart, lungs, liver, pancreas, spleen, stomach, small intestine, kidneys, bladder, testes, muscle, bone, adrenal gland, and blood. These organs were placed into glass tubes and weighed. A 2480 Wizard automatic gamma counter (PerkinElmer, USA) was used to determine how much radioactivity was reserved in them. A percentage of the %ID/g was shown to indicate the radioactivity concentration70.

4.10 Measurement of [18F]8 radio-metabolites in the brain and plasma of rats

SD rats (male, 7–8 weeks, 220–240 g) were injected with [18F]8 (37 MBq per animal) via the tail vein and decapitated at 5 and 30 min (n = 3, each time point) later. The blood and brain were collected and processed for analysis. The rat brain was homogenized in 50% CH3CN on ice and then centrifuged for 2.5 min at 4 °C (14,500×g). All blood samples were centrifuged at 6000 rpm for 5 min to get the upper plasma. The plasma samples were extracted and placed in CH3CN to sediment protein by centrifugation at 14,000 rpm for 3 min V1A-2303 standard was added to the above supernatant, and the mixture was separated into glass tubes at intervals of 30 s using semipreparative HPLC. Analyses were performed at room temperature (column OSAKA SODA CAPCELL PAK C18 UG80 5 μm, 10 mm × 250 mm) using a mobile phase of CH3CN/H2O (v/v, 50/50) containing 0.1% Et3N and at 3.0 mL/min. The radioactivity of separated samples was detected with the WIZARD2 2480 automatic gamma counter (PerkinElmer, USA). The unmetabolized [18F]8 parent fraction was determined as the ratio of the sum of radioactivity in fractions containing the parent compound to the total amount of radioactivity collected.

4.11 In vivo PET brain imaging of mice under baseline and P-glycoprotein (P-gp) blocking conditions

Each mouse was anesthetized by 2% isoflurane inhalation and an intravenous catheter was inserted into the tail vein. The radiotracer [18F]8 (ca. 5 MBq/150–200 μL) was injected into the mouse via the preinstalled catheter, and the 60 min dynamic acquisition of PET signals in the mouse brain was started at the same time. PET/CT imaging experiments were performed on an IRIS small animal PET/CT imaging system (Inviscan SAS, Strasbourg, France). To study the influence of P-glycoprotein (P-gp) on the brain uptake of the tracer, mice received an intravenous injection of cyclosporin (25 mg/kg) in saline (300 μL) containing 10% ethanol and 5% Tween® 80 at 15 min via the tail vein catheter before the tracer injection.

PET data were reconstructed with a three-dimensional ordered-subset expectation-maximizaton (3D-OSEM) algorithm with a Monte-Carlo based accurate detector model. PET images were then summed and co-registered with the mouse brain MRI template by PMOD software (version 4.1). The time‒activity curve (TAC) was acquired from the volume of interest (VOI) in the whole brain. The radioactivity was decay-corrected to the injection time and expressed as the standardized uptake value (SUV) which was normalized to the injected radioactivity and body weight. The area under the time–activity curve (AUC) was also calculated using the SUV on the TAC from 0 to 60 min.

4.12 PET imaging studies of [18F]V1A-2303 in monkey

Before the PET imaging study, rhesus monkeys were restrained from food for 12–15 h. Three male rhesus monkeys (weight range 6–7 kg) underwent a PET scan. The monkey lay on the scanning bed in the supine position and was anesthetized with a mixture of isoflurane and oxygen. After that, the monkey received a saline solution of [18F]V1A-2303 (111–148 MBq) and was followed by a dynamic PET scan of the head for 90 min. For blockade, 3 mg/kg of V1a receptor antagonist, balovaptan, was injected 5 min before [18F]V1A-2303.

To get accurate anatomical orientation of the monkey brain, each monkey was scanned by 3.0 T discovery MR 750 (GE Discovery 750, Milwaukee, USA) with a 3D Bravo T1 sequence (ketamine was injected intramuscularly into a rhesus monkey to make it stay anesthetized during the scan). Then, each monkey has its own T1-weighted MR image of brain as a template to co-register with PET.

Co-registration of PET and T1-weighted MR anatomical images was performed based on the literature method71. In this study, 3D T1-weighted MR images were segmented to determine the specific brain regions. We calculated the SUV based on the volumes of different brain regions.

4.13 Whole blood, plasma activity, and parent fraction of [18F]V1A-2303

A total of 25 arterial blood samples were collected during the 100 min PET scan of NHPs at 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 90, 105, 120, 150, 180, 210 and 240 s followed by 10, 20, 30, 45, 60, 75 and 90 min (2.0 mL for 2, 4, 10, 30, 60 and 90 min; the rest are 0.5 mL). The blood samples were quickly transferred to centrifuge tubes that had been rinsed with iced heparin and centrifuged (5 min, 6000 rpm, 4 °C) to obtain plasma. Then, we measured the radioactivity of each 100 μL of plasma and whole blood to get time–activity curves. A radioactive decay correction had been applied to all data from the injection time. We used the metabolite-corrected arterial plasma curve as input function. In order to analyze radio-metabolites, the upper plasma (collected at 2, 4, 10, 30, 60, and 90 min) was deproteinized with acetonitrile by centrifugation (3 min, 14,000 rpm, 4 °C). V1A-2303 standard was added to the above supernatant, and the mixture was separated into glass tubes at intervals of 30 s using semipreparative HPLC. The radioactivity of separated samples was detected with the WIZARD2 2480 automatic gamma counter (PerkinElmer, USA). [18F]V1A-2303 parent fraction was computed as a percent of the overall radioactivity (metabolites and parent).

4.14 Image data analysis

The T1-weighted MR anatomical image of the individual monkey, obtained using a 3.0 T discovery MR 750, was co-registered with the PET image using PMOD 4.1 (PMOD technology, Switzerland) software. This was done to analyze the imaging data within specific VOIs and to generate regional TACs. Regional VT and the kinetic rate constants (K) were calculated for the extracted TACs by reversible one-(1T) and two-(2T) tissue compartment model and Logan graphical analysis with the metabolite-corrected arterial PL input function.

4.15 VT time stability analysis

To assess the temporal stability of VT, data were progressively trimmed from 100 to 60 min and the corresponding VT values were normalized to the end-of-scan value at 100 min. VT was estimated from each truncated data set and normalized to the VT estimated from the full data set. Normalized VT that stabilizes within the range of 90%–110% is considered acceptable.

4.16 Statistical analysis

The AIC was employed to determine the optimal compartment model72, with lower AIC values indicating greater model parsimony. Furthermore, Pearson correlation analysis was conducted to assess the relationships between VT estimated by the 2TCM and the Logan Plot. Statistical significance was defined as a P-value less than 0.05. GraphPad Prism (version 9.5.1) was utilized for all statistical analyses. Continuous variables were reported as mean ± standard deviation (SD).

Acknowledgments

This work was financially supported by the 10.13039/501100001809 National Natural Science Foundation of China (Nos. 82071974 , 82102107 , and 82371998 ), the Science and Technology Program of Guangzhou, China (Nos. 202206010106 and 2023A04J1921 ), and the Guangdong Science and Technology Planning Project, China (2022A0505050042 ).

Author contributions

Junqi Hu: Investigation, Writing – original draft. Yinlong Li: Conceptualization, Data curation. Chenchen Dong: Data curation, Formal analysis, Validation. Huiyi Wei: Formal analysis, Methodology. Kai Liao: Validation. Junjie Wei: Resources. Chunyu Zhao: Formal analysis. Ahmad Chaudhary: Data curation. Jiahui Chen: Formal analysis. Hao Xu: Funding acquisition, Conceptualization. Ke Zhong: Writing – review & editing. Steven H. Liang: Supervision, Writing – review & editing. Lu Wang: Funding acquisition, Supervision. Weijian Ye: Funding acquisition, Supervision, Writing – original draft, Writing – review & editing.

Conflicts of interest

The authors declare no conflicts of interest.

Appendix A Supplementary information

The following is the Supporting Information to this article:Multimedia component 1

Multimedia component 1

Peer review under the responsibility of Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences.

Appendix A Supporting information to this article can be found online at https://doi.org/10.1016/j.apsb.2024.05.033.
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References

1 Sparapani S. Millet-Boureima C. Oliver J. Mu K. Hadavi P. Kalostian T. The biology of vasopressin Biomedicines 9 2021 89 33477721
2 Aikins A.O. Nguyen D.H. Paundralingga O. Farmer G.E. Shimoura C.G. Brock C. Cardiovascular neuroendocrinology: emerging role for neurohypophyseal hormones in pathophysiology Endocrinology 162 2021 bqab082
3 Koshimizu T.A. Nakamura K. Egashira N. Hiroyama M. Nonoguchi H. Tanoue A. Vasopressin V1a and V1b receptors: from molecules to physiological systems Physiol Rev 92 2012 1813 1864 23073632
4 Koshimizu T.A. Nasa Y. Tanoue A. Oikawa R. Kawahara Y. Kiyono Y. V1a vasopressin receptors maintain normal blood pressure by regulating circulating blood volume and baroreflex sensitivity Proc Natl Acad Sci U S A 103 2006 7807 7812 16682631
5 Fujiwara Y. Tanoue A. Tsujimoto G. Koshimizu T.A. The roles of V1a vasopressin receptors in blood pressure homeostasis: a review of studies on V1a receptor knockout mice Clin Exp Nephrol 16 2011 30 34 22038263
6 Fernández-Varo G. Oró D. Cable E.E. Reichenbach V. Carvajal S. de la Presa B.G. Vasopressin 1a receptor partial agonism increases sodium excretion and reduces portal hypertension and ascites in cirrhotic rats Hepatology 63 2015 207 216 26403564
7 Randesi M. Levran O. Correa da Rosa J. Hankins J. Rule J. Kreek M.J. Association of variants of arginine vasopressin and arginine vasopressin receptor 1a with severe acetaminophen liver injury Cell Mol Gastroenter 3 2017 500 505
8 Zhao N. Peacock S.O. Lo C.H. Heidman L.M. Rice M.A. Fahrenholtz C.D. Arginine vasopressin receptor 1a is a therapeutic target for castration-resistant prostate cancer Sci Transl Med 11 2019 eaaw4636
9 Liu H. Yu L. Yang L. Green M.S. Vasoplegic syndrome: an update on perioperative considerations J Clin Anesth 40 2017 63 71 28625450
10 Fernández-Varo G. Jiménez W. Cable E. Ginès P. Harris G. Bukofzer S. Partial vasopressin 1a receptor agonism reduces portal hypertension and hyperaldosteronism and induces a powerful diuretic and natriuretic effect in rats with cirrhosis and ascites Biomed Pharmacother 165 2023 115116
11 Winslow J.T. Insel T.R. Neuroendocrine basis of social recognition Curr Opin Neurobiol 14 2004 248 253 15082332
12 Rauen K. Trabold R. Brem C. Terpolilli N.A. Plesnila N. Arginine vasopressin V1a receptor-deficient mice have reduced brain edema and secondary brain damage following traumatic brain injury J Neurotrauma 30 2013 1442 1448 23441636
13 Dumais K.M. Veenema A.H. Vasopressin and oxytocin receptor systems in the brain: sex differences and sex-specific regulation of social behavior Front Neuroendocrinol 40 2016 1 23 25951955
14 Yang C. Zhang X. Gao J. Wang M. Yang Z. Arginine vasopressin ameliorates spatial learning impairments in chronic cerebral hypoperfusion via V1a receptor and autophagy signaling partially Transl Psychiat 7 2017 e1174
15 Dantzer R. Bluthe R.M. Koob G.F. Le Moal M. Modulation of social memory in male rats by neurohypophyseal peptides Psychopharmacology 91 1987 363 368 3104959
16 Everts H.G. Koolhaas J.M. Differential modulation of lateral septal vasopressin receptor blockade in spatial learning, social recognition, and anxiety-related behaviors in rats Behav Brain Res 99 1999 7 16 10512567
17 Engelmann M. Landgraf R. Microdialysis administration of vasopressin into the septum improves social recognition in Brattleboro rats Physiol Behav 55 1994 145 149 8140159
18 Landgraf R. Gerstberger R. Montkowski A. Probst J.C. Wotjak C.T. Holsboer F. V1 vasopressin receptor antisense oligodeoxynucleotide into septum reduces vasopressin binding, social discrimination abilities, and anxiety-related behavior in rats J Neurosci 15 1995 4250 4258 7790909
19 Hammock E.A.D. Lim M.M. Nair H.P. Young L.J. Association of vasopressin 1a receptor levels with a regulatory microsatellite and behavior Genes Brain Behav 4 2005 289 301 16011575
20 Bielsky I.F. Hu S.-B. Ren X. Terwilliger E.F. Young L.J. The V1a vasopressin receptor is necessary and sufficient for normal social recognition: a gene replacement study Neuron 47 2005 503 513 16102534
21 Veenema A.H. Bredewold R. De Vries G.J. Vasopressin regulates social recognition in juvenile and adult rats of both sexes, but in sex- and age-specific ways Horm Behav 61 2012 50 56 22033278
22 Kim S.J. Young L.J. Gonen D. Veenstra-VanderWeele J. Courchesne R. Courchesne E. Transmission disequilibrium testing of arginine vasopressin receptor 1A (AVPR1A) polymorphisms in autism Mol Psychiatry 7 2002 503 507 12082568
23 Yirmiya N. Rosenberg C. Levi S. Salomon S. Shulman C. Nemanov L. Association between the arginine vasopressin 1a receptor (AVPR1a) gene and autism in a family-based study: mediation by socialization skills Mol Psychiatry 11 2006 488 494 16520824
24 Yang S.Y. Cho S.C. Yoo H.J. Cho I.H. Park M. Yoe J. Family-based association study of microsatellites in the 5′ flanking region of AVPR1A with autism spectrum disorder in the Korean population Psychiatry Res 178 2010 199 201 20452058
25 Tansey K.E. Hill M.J. Cochrane L.E. Gill M. Anney R.J. Gallagher L. Functionality of promoter microsatellites of arginine vasopressin receptor 1A (AVPR1A): implications for autism Mol Autism 2 2011 3 21453499
26 Meyer-Lindenberg A. Domes G. Kirsch P. Heinrichs M. Oxytocin and vasopressin in the human brain: social neuropeptides for translational medicine Nat Rev Neurosci 12 2011 524 538 21852800
27 Bielsky I.F. Hu S.-B. Szegda K.L. Westphal H. Young L.J. Profound impairment in social recognition and reduction in anxiety-like behavior in vasopressin V1a receptor knockout mice Neuropsychopharmacol 29 2003 483 493
28 Sakakibara M. Felix-Ortiz A.C. Febo M. Gestational valproate alters BOLD activation in response to complex social and primary sensory stimuli PLoS One 7 2012 e37313
29 Umbricht D. del Valle Rubido M. Hollander E. McCracken J.T. Shic F. Scahill L. A single dose, randomized, controlled proof-of-mechanism study of a novel vasopressin 1a receptor antagonist (RG7713) in high-functioning adults with autism spectrum disorder Neuropsychopharmacol 42 2016 1914 1923
30 Weiss A. Wilson V.A.D. Hopkins W.D. Early social rearing, the V1A arginine vasopressin receptor genotype, and autistic traits in chimpanzees Autism Res 14 2021 1843 1853 34089305
31 Kimi S. Maiti R. Srinivasan A. Mishra B.R. Hota D. Efficacy and safety of V1a receptor antagonists in autism spectrum disorder: a meta-analysis Int J Dev Neurosci 84 2023 3 13 37641183
32 László K. Vörös D. Correia P. Fazekas C.L. Török B. Plangár I. Vasopressin as possible treatment option in autism spectrum disorder Biomedicines 11 2023 2603 37892977
33 Glavaš M. Gitlin-Domagalska A. Dębowski D. Ptaszyńska N. Łęgowska A. Rolka K. Vasopressin and its analogues: from natural hormones to multitasking peptides Int J Mol Sci 23 2022 3068 35328489
34 Baska F. Bozó É. Patócs T. Vasopressin receptor antagonists: a patent summary (2018‒2022) Expert Opin Ther Pat 33 2023 385 395 37226495
35 Yamamura Y. Ogawa H. Chihara T. Kondo K. Onogawa T. Nakamura S. OPC-21268, an orally effective, nonpeptide vasopressin V1 receptor antagonist Science 252 1991 572 574 1850553
36 Serradeil-Le Gal C. Wagnon J. Garcia C. Lacour C. Guiraudou P. Christophe B. Biochemical and pharmacological properties of SR 49059, a new, potent, nonpeptide antagonist of rat and human vasopressin V1a receptors J Clin Investig 92 1993 224 231 8392086
37 Fabio K.M. Guillon C.D. Lu S.-F. Heindel N.D. Brownstein M.J. Lacey C.J. Pharmacokinetics and metabolism of SRX246: a potent and selective vasopressin 1a antagonist J Pharm Sci 102 2013 2033 2043 23471831
38 Brownstein M.J. Simon N.G. Long J.D. Yankey J. Maibach H.T. Cudkowicz M. Safety and tolerability of SRX246, a vasopressin 1a antagonist, in irritable huntington's disease patients—a randomized phase 2 clinical trial J Clin Med 9 2020 3682 33207828
39 Lago T.R. Brownstein M.J. Page E. Beydler E. Manbeck A. Beale A. The novel vasopressin receptor (V1aR) antagonist SRX246 reduces anxiety in an experimental model in humans: a randomized proof-of-concept study Psychopharmacology 238 2021 2393 2403 33970290
40 Maibach H.T. Brownstein M.J. Hersch S.M. Anderson K.E. Itzkowitz D.E. Damiano E.M. The vasopressin 1a receptor antagonist SRX246 reduces aggressive behavior in Huntington's disease J Pers Med 12 2022 1561 36294700
41 Ratni H. Rogers-Evans M. Bissantz C. Grundschober C. Moreau J.L. Schuler F. Discovery of highly selective brain-penetrant vasopressin 1a antagonists for the potential treatment of autism via a chemogenomic and scaffold hopping approach J Med Chem 58 2015 2275 2289 25654260
42 Schnider P. Bissantz C. Bruns A. Dolente C. Goetschi E. Jakob-Roetne R. Discovery of balovaptan, a vasopressin 1a receptor antagonist for the treatment of autism spectrum disorder J Med Chem 63 2020 1511 1525 31951127
43 Bolognani F. del Valle Rubido M. Squassante L. Wandel C. Derks M. Murtagh L. A phase 2 clinical trial of a vasopressin V1a receptor antagonist shows improved adaptive behaviors in men with autism spectrum disorder Sci Transl Med 11 2019 eaat7838
44 Hollander E. Jacob S. Jou R. McNamara N. Sikich L. Tobe R. Balovaptan vs placebo for social communication in childhood autism spectrum disorder JAMA Psychiatry 79 2022 760 769 35793101
45 Jacob S. Veenstra-VanderWeele J. Murphy D. McCracken J. Smith J. Sanders K. Efficacy and safety of balovaptan for socialisation and communication difficulties in autistic adults in North America and Europe: a phase 3, randomised, placebo-controlled trial Lancet Psychiat 9 2022 199 210
46 Fabio K. Guillon C. Lacey C.J. Lu S.F. Heindel N.D. Ferris C.F. Synthesis and evaluation of potent and selective human V1a receptor antagonists as potential ligands for PET or SPECT imaging Bioorg Med Chem 20 2012 1337 1345 22249122
47 Naik R. Valentine H. Hall A. Mathews W.B. Harris J.C. Carter C.S. Development of a radioligand for imaging V1a vasopressin receptors with PET Eur J Med Chem 139 2017 644 656 28843869
48 Haider A. Xiao Z. Xia X. Chen J. Van R.S. Kuang S. Development of a triazolobenzodiazepine-based PET probe for subtype-selective vasopressin 1A receptor imaging Pharmacol Res 173 2021 105886
49 Rong J. Haider A. Jeppesen T.E. Josephson L. Liang S.H. Radiochemistry for positron emission tomography Nat Commun 14 2023 3257 37277339
50 Abbott N.J. Prediction of blood–brain barrier permeation in drug discovery from in vivo, in vitro and in silico models Drug Discov Today Technol 1 2004 407 416 24981621
51 Kunwittaya S. Nantasenamat C. Treeratanapiboon L. Srisarin A. Isarankura-Na-Ayudhya C. Prachayasittikul V. Influence of logBB cut-off on the prediction of blood‒brain barrier permeability Biomed Appl Technol J 1 2013 16 34
52 Wager T.T. Hou X. Verhoest P.R. Villalobos A. Moving beyond rules: the development of a central nervous system multiparameter optimization (CNS MPO) approach to enable alignment of druglike properties ACS Chem Neurosci 1 2010 435 449 22778837
53 Łabuzek K. Suchy D. Gabryel B. Bielecka A. Liber S. Okopień B. Quantification of metformin by the HPLC method in brain regions, cerebrospinal fluid and plasma of rats treated with lipopolysaccharide Pharmacol Rep 62 2010 956 965 21098880
54 Deng X. Rong J. Wang L. Vasdev N. Zhang L. Josephson L. Chemistry for positron emission tomography: recent advances in 11C-, 18F-, 13N-, and 15O-labeling reactions Angew Chem Int Ed 58 2019 2580 2605
55 Pettibone D.J. Kishel M.T. Woyden C.J. Clineschmidt B.V. Bock M.G. Freidinger R.M. Radioligand binding studies reveal marked species differences in the vasopressin V1 receptor of rat, rhesus and human tissues Life Sci 50 1992 1953 1958 1593923
56 Gal C.S.L. Raufaste D. Marty E. Garcia C. Maffrand J.P. Le Fur G. Autoradiographic localization of vasopressin V1a receptors in the rat kidney using [3H]-SR 49059 Kidney Int 50 1996 499 505 8840278
57 Vågnes Ø.B. Hansen F.H. Christiansen R.E.F. Gjerstad C. Iversen B.M. Age-dependent regulation of vasopressin V1a receptors in preglomerular vessels from the spontaneously hypertensive rat Am J Physiol-renal 286 2004 F997 F1003
58 Smith C.J.W. Poehlmann M.L. Li S. Ratnaseelan A.M. Bredewold R. Veenema A.H. Age and sex differences in oxytocin and vasopressin V1a receptor binding densities in the rat brain: focus on the social decision-making network Brain Struct Funct 222 2016 981 1006 27389643
59 Grazzini E. Breton C. Derick S. Andres M. Raufaste D. Rickwaert F. Vasopressin receptors in human adrenal medulla and pheochromocytoma1 J Clin Endocr Metab 84 1999 2195 2203 10372731
60 Uchida Y. Ohtsuki S. Katsukura Y. Ikeda C. Suzuki T. Kamiie J. Quantitative targeted absolute proteomics of human blood–brain barrier transporters and receptors J Neurochem 117 2011 333 345 21291474
61 Tan Z. Haider A. Zhang S. Chen J. Wei J. Liao K. Quantitative assessment of translocator protein (TSPO) in the non-human primate brain and clinical translation of [18F]LW223 as a TSPO-targeted PET radioligand Pharmacol Res 189 2023 106681
62 Ottoy J. Verhaeghe J. Niemantsverdriet E. Wyffels L. Somers C. De Roeck E. Validation of the semiquantitative static suvr method for 18F-AV45 PET by pharmacokinetic modeling with an arterial input function J Nucl Med 58 2017 1483 1489 28336779
63 Yan X. Siméon F.G. Liow J.-S. Morse C.L. Montero Santamaria J.A. Jenkins M. In vivo evaluation of a novel 18F-labeled PET radioligand for translocator protein 18 kDa (TSPO) in monkey brain Eur J Nucl Med Mol Imaging 50 2023 2962 2970 37249618
64 Maher B.S. Vladimirov V.I. Latendresse S.J. Thiselton D.L. McNamee R. Kang M. The AVPR1A gene and substance use disorders: association, replication, and functional evidence Biol Psychiatry 70 2011 519 527 21514569
65 Nishina K. Takagishi H. Takahashi H. Sakagami M. Inoue-Murayama M. Association of polymorphism of arginine-vasopressin receptor 1a (AVPR1a) gene with trust and reciprocity Front Hum Neurosci 13 2019 230 31354450
66 Tanaka H. Nishina K. Shou Q. Takahashi H. Sakagami M. Matsuda T. Association between arginine vasopressin receptor 1A (AVPR1A) polymorphism and inequity aversion Proc Biol Sci 290 2023 20230378
67 Yung-Chi C. Prusoff W.H. Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction Biochem Pharmacol 22 1973 3099 3108 4202581
68 Gartzke D. Fricker G. Establishment of optimized MDCK cell lines for reliable efflux transport studies J Pharm Sci 103 2014 1298 1304 24532159
69 Tournier N. Cisternino S. Peyronneau M.A. Goutal S. Dolle F. Scherrmann J.M. Discrepancies in the P-glycoprotein-mediated transport of 18F-MPPF: a pharmacokinetic study in mice and non-human primates Pharm Res 29 2012 2468 2476 22644589
70 Su L. Dalby K.S. Luehmann H. Elkassih S.A. Cho S. He X. Ultrasmall, elementary and highly translational nanoparticle X-ray contrast media from amphiphilic iodinated statistical copolymers Acta Pharm Sin B 13 2023 1660 1670 37139426
71 McLaren D.G. Kosmatka K.J. Oakes T.R. Kroenke C.D. Kohama S.G. Matochik J.A. A population-average MRI-based atlas collection of the Rhesus macaque Neuroimage 45 2009 52 59 19059346
72 Akaike H. A new look at the statistical model identification IEEE Trans Automat Control 19 1974 716 723
