Synthesis and Biological Evaluation of [18F]FECNT-d as a Novel PET Agent for Dopamine Transporter Imaging
Shanshan Cao,1 Jie Tang,2 Chunyi Liu,2 Yi Fang,2 Linyang Ji,1 Yingjiao Xu,2 Zhengping Chen 1,2 Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy, Xuzhou Medical University, Xuzhou, 221004, China
Abstract
Purpose: The dopamine transporter (DAT) is a marker of the occurrence and development of Parkinson’s disease (PD) and other diseases with nigrostriatal degeneration. 2β-Carbomethoxy-3β(4-chlorophenyl)-8-(2-[18F]-fluoroethyl)nortropane ([18F]FECNT), an 18F-labelled tropane derivative, was reported to be a useful positron-emitting probe for DAT. However, the rapid formation of brainpenetrating radioactive metabolites is an impediment to the proper quantitation of DAT in PET studies with [18F]FECNT. Deuterium-substituted analogues have presented better in vivo stability to reduce metabolites. This study aimed to synthesize a deuterium-substituted DAT radiotracer, [18F]FECNT-d4, and to make a preliminary investigation of its properties as a DAT tracer in vivo. Procedures: The ligand [18F]FECNT-d4 was obtained by one-step radiolabelling reaction. The lipophilicity was measured by the shake-flask method. Binding properties of [18F]FECNT-d4 were estimated by in vitro binding assay, biodistribution, and microPET imaging in rats. In vivo stability of [18F]FECNT-d4 was estimated by radio-HPLC.
Results: [18F]FECNT-d4 was synthesized at an average activity yield of 46 ± 17 % (n = 15) and the molar activity was 67 ± 12 GBq/μmol. The deuterated tracer showed suitable lipophilicity and the ability to penetrate the blood-brain barrier (brain uptake of 1.72 % ID at 5 min). [18F]FECNT-d4 displayed a high binding affinity for DAT comparable to that of [18F]FECNT in rat striatum homogenates. Biodistribution results in normal rats showed that [18F]FECNT-d4 exhibited a higher ratio of the target to non-target (striatum/cerebellum) at 15 min post administration (5.00 ± 0.44 vs 3.84 ± 0.24 for [18F]FECNT-d4 vs [18F]FECNT). MicroPET imaging studies of [18F]FECNT-d4 in normal rats showed that the ligand selectively localized to DAT-rich striatal regions and the accumulation could be blocked with DAT inhibitor. Furthermore, in the unilateral PD model rat, a significant reduction of the signal was found in the lesioned side relative to the unlesioned side. Striatal standardized uptake value of [18F]FECNT-d4 remained ~4.02 in the striatum between 5 and 20 min, whereas that of [18F]FECNT fell rapidly from 4.11 to 2.95. Radio-HPLC analysis of the plasma demonstrated better in vivo stability of [18F]FECNT-d4 than [18F]FECNT.
Conclusion: The deuterated compound [18F]FECNT-d4 may serve as a promising PET imaging agent to assess DAT-related disorders.
Key words: DAT, MicroPET imaging, 18F, Deuterium, In vivo stability
Introduction
The dopamine transporter (DAT) plays almost the sole executor of dopamine reutilization which is vitally correlated with Parkinson’s disease (PD), and other neurodegenerative diseases [1, 2]. The dramatic loss of dopaminergic neurons in early-stage PD, found in previous clinicopathologic studies, suggests that the degeneration of this region begins before motor symptoms [3, 4]. As widely expressed in dopaminergic neurons, DAT is an important indicator for the diagnosis of early PD. Therefore, potential radioligands for image DAT with positron emission tomographic (PET) may aid diagnosis and treatment of PD by providing probes to estimate the density of DAT in specific brain regions.
Over the decades, researchers have developed several DAT radiotracers for PET imaging, 11C-labelled ligands such as [11C]PE2I [5] and [11C]CFT [6]. However, 11C has a very short half-life (20 min); 18F-labelled ligands with a longer half-life (109 min) would be more practical for widespread use, such as [18F]FP-CMT [7], [18F]FE-PE2I [8], [18F]LBT-999 [9], and [18F]FECNT (2β-carbomethoxy-3β(4-chlorophenyl)-8-(2-[18F]fluoroethyl)nortropane) [10]. [18F]FECNT has 25- and 156-fold selectivity for DAT (Ki = 1.5 nM) more than for serotonin transporter and noradrenaline transporter, respectively [10]. Davis et al. reported that PET imaging with [18F]FECNT has favorable kinetics in humans and could distinguish DAT deficits between Parkinson’s patients and healthy individuals [11]. However, it is found that [18F]FECNT, which has the 18F on the N-alkyl group, tends to be de-alkylated by cytochrome P450 enzyme to form radioactive metabolites, namely [18F]fluoroacetaldehyde, [18F]fluoroethanol, and [18F]fluoroacetatic acid [12]. These metabolites can cross the blood-brain barrier (BBB) and distribute in the whole brain, confounding quantitative measurement of DAT. To accurately quantify the uptake and binding of [18F]FECNT to DAT, Nye and colleagues raised an arterial input model of [18F]FECNT uptake for DAT density quantification in humans [13]. However, this protocol required a 180-min PET scan and a series of arterial blood samples during imaging, which might be not convenient enough for clinical routine application. Nevertheless, this N-dealkylation metabolism pathway is not unique to [18F]FECNT and has also been observed in other ligands with high affinity and selectivity for DAT such as [18F]FE-PE2I [14] and [18F]LBT-999 [15].
In recent years, a great deal of work has revealed the deuterium isotope effect on the biological reaction [16, 17]. Due to the cleavage rate of a carbon-deuterium (C–D) bond being about 6–7 times slower than that of a carbon-hydrogen (C–H) bond, the deuterium-substitution at easily metabolizable positions of the molecules can help to improve the metabolic stability of active pharmaceuticals [18, 19]. Recently, a deuterated form of tetrabenazine, SD-809, has been approved by the FDA to treat tardive dyskinesia [20]. Another example is CTP-656, a deuterated ivacaftor structure, which overcame many defects of the original drug such as short half-life and high clinical dosage [21]. Recently, the deuterium substitution strategy has also been applied to radiopharmaceutical for PET imaging. For example, [18F]FE-(+)-DTBZ-d4 [22] and [18F]MNI-659-d2 [23] were employed as radiotracers for vesicular monoamine transporter 2 (VMAT2) and phosphodiesterase 10A (PDE 10A), respectively.
In this study, we designed and synthesized a deuterated F-labelled analogue [ F]FECNT-d4 (Fig. 1), aiming to improve upon the in vivo metabolic stability of [18F]FECNT. Herein, we performed binding affinity, lipophilicity, autoradiography, biodistribution, and PET imaging of [18F]FECNT-d4. Our findings of excellent stability in vivo and high specific binding properties of [18F]FECNT-d4 imply that the deuterated tracer may be an attractive PET imaging agent for DAT.
Materials and Methods
General
All of the organic solvents and chemical reagents were obtained from Aladdin (China), J&K Scientific (China), Sigma-Aldrich (China), and used directly without further purification. FECNT and CFT were synthesized according to previously published papers [24, 25]. Kryptfix222 (K222, 98.0 %) was purchased from ABX (Germany). Sep-Pak C18 cartridges and Sep-Pak lightweight QMA (QMA) were purchased from Waters (USA). Analytical highperformance liquid chromatography (HPLC) system was used to evaluate radiochemical purity (RCP) and the radiochemical yield (RCY) of [18F]FECNT and[18F]FECNT-d4. Analytical high-performance liquid chromatography (HPLC) system consists of Waters 1525 HPLC pump, 2489 UV/Visible Detector (Waters, USA), and radiometric 610TR detector (PerkinElmer, USA). The analytical conditions were as follows: analytical column (C18, 5 μm, 4.6 × 150 mm, Phenomenex, Gemini, USA), 1.0 mL/min, UV detection at 220 nm, method A: H2O/ CH3CN/Et3N (40/60/0.1, v/v/v); method B: H2O/CH3CN/ Et3N (38/62/0.1, v/v/v). Semi-prepared HPLC (semi-prep HPLC) consists of Waters 1525 HPLC pump, 2489 UV/Visible Detector (Waters, USA), and flow-count radioactivity detector (Biscan, USA). The semi-prep HPLC system was used to separate and purify crude products. The preparation conditions (method C) were as follows: semiprep column (C18, 5 μm, 10 × 250 mm, Waters, XBridge, USA), 5.0 mL/min, UV detection at 220 nm, H2O/CH3CN/ Et3N (45/55/0.1, v/v/v). The chemical structure of [18F]FECNT-d4 was identified by co-injected with the nonlabelled standard compound.
The human neuroblastoma (SH-SY5Y) cell line was purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China). The cells were cultured inDMEM: F12 medium (containing 1 % penicillinstreptomycin, 1 % gluta-max, 15 % fetal bovine serum) and kept under an atmosphere of 5 % CO2 at 37 °C.
Male Sprague-Dawley (SD, 220−260 g) rats and Institute of Cancer Research mice (ICR, 20−30 g) were provided by Vital River Laboratory Animal Technology Co. Ltd. (China). The striatal unilateral 6-hydroxydopamine (6OHDA) lesion model was performed with saline containing 16 μg (4 μg/μl) 6-OHDA, at the coordinates: AP: −4.0 mm (Bregma), ML: −1.4 mm (midline), DV: −7.5 mm (hole) [26]. All the animals were housed in clean cages with free access to food and water with a 12-h light-dark cycle and then tested. All animal experiment procedures were approved by the Animal Care and Ethics Committee of Jiangsu Institute of Nuclear Medicine.
A mixture of (7) (0.5 mmol, 1.0 equiv.) and methanesulfonic anhydride (2.2 mmol, 4.4 equiv.) in freshly dried dichloromethane (2 mL) was stirred at 30 °C for 3 days under nitrogen. The chemical purity of (8) was monitored by the analytical HPLC under the following condition (H2O/ CH3CN/TFA, 65/35/0.2, v/v/v). After the reaction was complete, the solvent was removed under reduced pressure. The residue was dissolved in 1 mL dichloromethane and washed with 2 mL ether 5 times. Finally, the precipitate was obtained by 99 % yield as a white solid (8) under reduced pressure. 1H NMR (400 MHz, CDCl3) δ 9.21 (s, 1H), 7.33–
The synthetic method of [18F]FECNT was referenced to the previous article [24]. The automatic radiosynthesis of [18F]FECNT-d4 was completed by the PET-MF-2 V-IT-1 (Beijing PET Technology, China) multifunctional synthesis module. First, [18F]fluoride (10 GBq) was captured on the QMA cartridge by the enriched [18O]H2O through cyclotron. Then 18F ion was transferred into the reaction tube from the irradiated QMA with a stock solution ((K222 (40 μmol), CH3CN (0.8 mL), K2CO3 (20 μmol), water (0.2 mL)). The mixture was dried at 105 °C for 2 min under nitrogen, and further dried twice at 105 °C with ultra-dry acetonitrile. After drying, cool to 40 °C, the precursor (8) (5 mg) dissolved in CH3CN (1 mL) was added to the reaction tube and kept at 90 °C for 20 min. Next, the reaction was quenched with H2O (0.5 mL) and the mixture was transferred to the semi-prepared HPLC (method C). After purification, the collected liquid was diluted with 10-fold water and the tracer [18F]FECNT-d4 was trapped using a Sep-Pak C18 light cartridge. Then, the product was eluted with 1 mL of ethanol from the C18 cartridge. The product was analyzed for quality control by radio-HPLC (method A).
In vitro Stability Assays
The stability of [18F]FECNT-d4 was assayed in phosphatebuffered solution (PBS) and fetal bovine serum (FBS). [18F]FECNT-d4 (37 MBq) was added to PBS (pH 7.4, 500 μL) and FBS (500 μL), respectively. The mixtures were incubated for 0,1,2,4,and6hat37°C.Theradiochemicalpuritywasrecorded by analytical radio-HPLC (method A) at each time point. For serum samples, acetonitrile (100 μL) was added to a serum sample(100μL)andtheproteinprecipitatewasremovedbyhighspeed centrifugation before purity analysis.
In vivo Stability Assays
Following the injection of [18F]FECNT-d4 or [18F]FECNT (20 MBq in 0.2 mL 10 % ethanol/saline) to the mice (n = 3), the blood samples were obtained from the mice’s tail at 5, 30, and 60 min. Each sample was collected in a test tube containing CH3CN (50 μL) with FECNT (20 μM). Male Sprague-Dawley rats (n = 3) were anesthetized with 2.5 % isoflurane in oxygen followed by intravenous injection of radioligand ([18F]FECNT-d4 or [18F]FECNT: 185 MBq in 0.5 mL 10 % ethanol/saline). At 5, 30, and 60 min after injection, three rats were sacrificed at each time point respectively. The striatum and cerebellum were rapidly removed. Then, each sample of isolated striatum, cerebellum, and plasma was homogenized in 0.5 mL acetonitrile containing 1.0 mg/ml FECNT, respectively. The cold FECNT was added in samples to improve the extraction efficiency. The homogenates were centrifuged at 12,000g for 5 min at 4 °C for deproteinization. After centrifugation, the radioactivity counts of the supernatant and the precipitate were measured with a γ-counter (2480 Wizard2, PerkinElmer, USA) separately to calculate the extraction efficiency of radioactive material. Finally, a portion of the resulting supernatant was injected into the analytical radioHPLC system (method B). On the HPLC chromatogram, the percent ratio was calculated as the peak area of the unchanged form to total peak area (corrected for decay) × 100 %.
Cellular Uptake and Blocking Assays
SH-SY5Y cells were seeded into 12-well plates (1.5 × 105 cells per well) and cultured at 37 °C overnight for cell adherence. During the experiment, the cells were assigned for the control group, CFT blocking group, and DTBZ blocking group. The control group was added with [18F]FECNT-d4 (18.5 KBq) in fresh medium (200 μL), the other two groups were added with [18F]FECNT-d4 and CFT (20 μM) or [18F]FECNT-d4 and DTBZ (20 μM). The cells were separately incubated at 37 °C for 15, 30, 60, 90, and 120 min. At each time point, the basic medium was extracted to a test tube, and the cells were rinsed once with ice-cold PBS. Then, the cells were lysed and collected with NaOH (1 M) resolution. The radioactivity counts of cell lysate and supernatant were measured by a γ-counter and calculated as % uptake. The cellular uptake values of [18F]FECNT-d4 (mean ± SD, n = 3) were calculated as following formula: [CPM (the cell lysate)] / [CPM (the cell lysate) + CPM (the medium)] × 100 %. The blocking percentage was calculated with [CPM (control) − CPM (with inhibitor)] / CPM (control)] × 100 %.
Determination of Lipophilicity
The octanol-water partition coefficient of [18F]FECNT-d4 and [18F]FECNT was determined at room temperature by the shake-flask method. [18F]FECNT-d4 or [18F]FECNT (18.5 KBq) was added into two-phase mixture of n-octanol (3 mL) and PBS (pH 7.4, 3 mL). After shaking for 5 min, the PBS and the n-octanol phase were separated via centrifugation. Then, the PBS (1 mL) solution and n-octanol (1 mL) were pipetted into test tubes, respectively. The radioactivity in noctanol and PBS were determined by a γ-counter. Subsequently, the remaining n-octanol (1 mL) was redistributed into a mixture containing 2 mL n-octanol and 3 mL PBS. The above experiment was repeated 5 times to obtain an average logP value. The logP values were calculated by the formula: logP = log(CPMn-octanol/CPMPBS). All measurements were tested in triplicate.
Competitive Binding Assays
The rats were decapitated under ether anesthesia. The striatum (ST) was quickly pulled out and homogenized in 50 mM Tris-HCl buffer (pH 7.4) containing 120 mM NaCl, 2 mM KCl, 1 mM CaCl2, and 1 mM MgCl2 through a high throughput group grinder. Each 100 μL of homogenate is incubated with [18F]FECNT (0.5–0.8 nM in buffer) and 100 μL of various concentrations of the test compound (FECNTd4 or FECNT, 10−11–10−7 M) in a final volume of 0.5 mL of buffer. Non-specific binding was determined by coincubation with 20 μM of CFT. These mixtures were incubated at room temperature for 30 min and terminated by rapid filtration using Whatman GF/C glass-fiber filters, followed by washing four times with ice-cold PBS. The radioactivity of the pellets bond with [18F]FECNT was measured using an automatic γ-counter. The 50 % inhibitory concentration (IC50) was calculated using the nonlinear least-square curve fitting with GraphPad Prism.
Biodistribution Studies
The rats were divided into 10 groups (n = 5) for the biodistribution studies. Each rat was injected with radioligand ([18F]FECNT-d4 or [18F]FECNT: 12 MBq in 0.5 ml 10 % ethanol/saline) through the tail vein, and they were executed by cervical dislocation at 5, 15, 30, 60, and 120 min under anesthesia. The whole brain (including the striatum, cerebellum (CB), hippocampus (HIP), cortex (CX)) and each organ (including the heart, liver, spleen, lung, kidney, stomach, small intestines, testis, bone, muscle, pancreas) were quickly dissected and weighed. In addition, blood (200 μL) was obtained from the carotid artery. These radioactive samples were counted by a γ-counter and the results were expressed as a percentage of the injected dose per organ (% ID) or a percentage of the injected dose per gram of wet tissue (% ID/g) by calibrating with 1 % initial dose counts (100-fold diluted of the injection).
Ex vivo Autoradiography
Normal and 6-OHDA model rats were respectively injected with 37 MBq [18F]FECNT-d4 via the tail vein and sacrificed at 30 min post injection. The brain of each rat was separated and frozen quickly. Coronal sections (20 μm) were cut on a cryostat and dried at room temperature. The dried sections were exposed to the imager plate for 45 min, and images were attained using Cyclone Plus Storage Phosphor System (Perkin Elmer, USA).
MicroPET Imaging
A high-resolution microPET system (Siemens Medical Solutions, Knoxville, TN, Germany) was used to monitor 0–120 min scanning of the rat brain in real time. The rat was placed on the bed of microPET and the brain position was fixed under isoflurane anesthesia (2.5 % in oxygen gas) during the experiment. Each rat (n = 5) was injected with 12 MBq of [18F]FECNT-d4 or [18F]FECNT (0.5 ml, 10 % ethanol/saline) through the tail vein. The scan results were acquired in 3 min per frame sequences to obtain 40 consecutive frames, and then images were reconstructed using the analytical filter back projection (FBP) method. Time activity curves (TACs) for tissue uptake were expressed as standard uptake values (SUVs). The injected radioactivity was corrected by time decay. To verify the specific binding of [18F]FECNT-d4 to DAT in vivo, a DAT-specific ligand CFT (1.0 mg/kg) was coinjected with [18F]FECNT-d4 into the tail vein and the rats were scanned from 10 to 40 min. Furthermore, the PD model rats were also scanned at 10–40 min after administration of [18F]FECNT-d4. All obtained images were processed and analyzed with ASIPro VM software; the regions of interest (ROIs) were plotted on summational images for a sum of all frames [28]. The binding potential (BPND) in the striatum was calculated by the simplified reference tissue method [29] using the TAC with the cerebellum as a reference region.
Results
Synthesis
The non-radioactive compound FECNT-d4 was synthesized with (6) in 38 % yield. The mesylate precursor (8) was obtained in 99 % yield by esterification (Scheme 1). Finally, [18F]FECNT-d4 was synthesized from (8) by one step in a multifunctional synthesis module. The crude product solution was clear and colorless, and the RCP was greater than 99.9 % after purification. The molar activity (Am) was 67 ± 12 GBq/μmol and the RCY was 46 ± 17 % (n = 15) at the end of [18F]FECNT-d4 synthesis.
In vitro Stability Assays
[18F]FECNT-d4 did not breakdown during its synthesis procedure. The HPLC analysis results showed satisfactory stability of [18F]FECNT-d4 for up to 6 h at 37 °C with RCP 9 98 % in PBS and 9 96 % in serum, respectively (Fig. 2).
In Vivo Stability Assays
To verify the in vivo stability of [18F]FECNT-d4 and [18F]FECNT, it was necessary to analyze the parent tracer content in mice or rats after injected with radiotracer using radio-HPLC. The analysis showed that the retention times in the HPLC diagram were approximately 2.2 min for polar radioactive metabolites and 7.5 min for parent radiotracer. For mice, after administration of [18F]FECNT-d4 or [18F]FECNT, polar metabolites of [18F]FECNT began to appear in plasma at 30 min post administration. More metabolites of [18F]FECNT were observed at 60 min, whereas only a little fraction of radiometabolites of [18F]FECNT-d4 were detected (Suppl. Fig. 1, see ESM). For rats, the parent tracer and radiolabelled metabolite fractions in brain tissues and plasma were described in Table 1.
Cellular Uptake and Blocking Assays
The cell uptake of [18F]FECNT-d4 was evaluated in SHSY5Y cells. The accumulation of [18F]FECNT-d4 in SHSY5Y cells reached 1.75 % uptake at 120 min. The binding specificity of [18F]FECNT was preliminarily evaluated using CFT- and DTBZ-treated cells. Compared to the control group, the accumulation of [18F]FECNT-d4 was inhibited ~66 % (0.60 % uptake) by an excess of CFT at 120 min (Suppl. Fig. 2, see ESM). All the time, the uptake of [18F]FECNT-d4 was not influenced by DTBZ.
Lipophilicity and Binding Affinity
As shown in Table 2, the logP7.4 values of [18F]FECNT-d4 and [18F]FECNT were found to be 2.37 and 2.25, respectively. The IC50 values of the two compounds exhibited in the single-digit nanomolar range (FECNT-d4, IC50 = 2.17 nM and FECNT, IC50 = 1.99 nM).
Biodistribution Studies
The biodistribution results of [18F]FECNT-d4 and [18F]FECNT at each time point (5, 15, 30, 60, and 120 min) in rats were shown in Fig. 3. After the rats were severally injected with [18F]FECNT-d4 and [18F]FECNT, the tracers were distributed primarily to the liver (15.81 ± 0.34 % ID vs 16.88 ± 1.46 % ID), lung (1.12 ± 0.17 % ID vs 1.93 ± 0.59 % ID), kidney (1.94 ± 0.08 % ID vs 1.82 ± 0.18 % ID), and brain (1.72 ± 0.10 % ID vs 1.66 ± 0.20 % ID), and lower accumulation in the bone, heart, small intestine, pancreas, and spleen. Brain distribution showed that the activity of [18F]FECNT-d4 in the striatum reached 2.03 ± 0.34 % ID/g at 5 min and retained at 2.06 ± 0.07 % ID/g until 30 min. The activity of [18F]FECNT decreased rapidly in the striatum from 5 min (2.51 ± 0.28 % ID/g) to 30 min (1.58 ± 0.19 % ID/g).
Ex vivo Autoradiography
The regional distribution of [18F]FECNT-d4 in rat brain was further assessed by ex vivo autoradiography. As shown in Fig. 4, [18F]FECNT-d4 was located substantially in the caudate putamen (CPu), substantial nigra (SN), and nucleus accumbens (NAc), with low concentration in the cerebellum and cortex.
MicroPET Imaging
To confirm in vivo biological activity of [18F]FECNT-d4, microPET imaging was performed with normal and PD model rats. As shown in Fig. 5, in normal rats, prominent radioactivity was found in the striatum and the concentration could be inhibited to the background level by CFT. Meanwhile, in 6-OHDA unilaterally lesioned brains, the concentration of [18F]FECNT-d4 was noticeably reduced in the lesioned side relative to the unlesioned side. Dynamic TACs showed that the radioactivity of [18F]FECNT-d4 in the striatum maintained high uptake with SUVs of 4.02 ± 0.45 at 5–20 min, and the SUVs of [18F]FECNT reached 4.11 ± 0.61 at 5 min and then declined rapidly. The volume of interest analysis by the Logan reference tissue method showed BPND of 1.95 ± 0.22 (mean ± SD, n = 5) for [18F]FECNT-d4 and 1.65 ± 0.51 (n = 5) for [18F]FECNT, respectively.
Discussion
In this study, we synthesized [18F]FECNT-d4 as a PET ligand for DAT based on the chemical structure of FECNT. The novel agent [18F]FECNT-d4 presents a better target to non-target ratio and in vivo stability than the previously used contrast agent [18F]FECNT. However, its application as a DAT radioligand in humans still needs further investigation. The present study provides the basis for future research.
To preliminarily assess [18F]FECNT-d4 bioactivity in vitro, we applied the radiotracer to the cellular uptake assay. The cellular uptake of [18F]FECNT-d4 showed a remarkable climbing tendency in 2 h, and the uptake of [18F]FECNT-d4 after pretreatment with the DAT inhibitor showed a 66 % tracer binding inhibition. However, the radioactivity did not decline when incubated with VMAT2 ligand. The results initially demonstrated its bioactivity and high selectivity for DAT (Suppl. Fig. 2, see ESM). Then, the binding affinity of FECNT-d4 or FECNT to DAT was determined using [18F]FECNT and rat striatum membrane homogenates. The results showed that FECNT-d4 exhibited a comparable affinity for DAT to FECNT in the nanomolar range [10], which implied that binding affinity was almost unaffected by hydrogen-deuterium substitution.
Lipophilicity is one of the factors limiting a drug’s ability to cross the BBB. In agreement with our hypothesis, the results of lipophilicity displayed that [18F]FECNT-d4 meets the criteria of appropriate logP value (2 G logP G 3.5) to cross the BBB [30]. This argument was also supported by the biodistribution of brain uptake. Biodistribution results showed that [18F]FECNT-d4 was rapidly distributed to the striatum but not retained in the regions without DAT. The ratio of target to non-target (ST/CB) at equilibrium can be used to evaluate specific binding in the target region of the brain. A comparative study of the biodistribution of [18F]FECNT-d4 and [18F]FECNT in rats displayed that the two tracers have different brain metabolic trends (Fig. 3a, b). The ST to CB ratio of [18F]FECNT-d4 was higher than [18F]FECNT at each point except at 5 min (Suppl. Table 2 and 4, see ESM). A slower elimination rate of [18F]FECNTd4 than [18F]FECNT was also found in other high uptake organs (Fig. 3c, d). These results indicated that [18F]FECNT-d4 improved the ratio of target to non-target and decelerated the drug’s degradation rate in vivo compared to [18F]FECNT. Besides, we found that the bone uptakes in biodistribution results were very low from 0 to 120 min for both [18F]FECNT-d4 and [18F]FECNT, suggesting the stability of [18F]fluoroethyl group with negligible in vivo defluorination [18, 31].
Then, the specific binding of [18F]FECNT-d4 to DAT in vivo was confirmed using ex vivo autoradiography and microPET imaging. Autoradiography in normal rats showed high accumulation of [18F]FECNT-d4 in the CPu, NAc, and SN, with abundant radioactivity in the symmetrical striata and low uptake in the CB and cortex. The binding specificity of [18F]FECNT-d4 was confirmed to be consistent with the known localization of DAT in rodent brains [32]. Besides, in unilateral PD model rats, an obvious reduction of [18F]FECNT-d4 uptake was found in the lesioned hemisphere relative to the unlesioned side (Fig. 4).
MicroPET imaging was performed on normal and PD model rats to further evaluate the potential of [18F]FECNTd4 for DAT tracing (Fig. 5a). The resulting images were well aligned with autoradiography which showed high tracer uptake in regions of DAT-rich striata. The sufficient brain uptake and rapid washout of non-target tissue of the tracer showed in dynamic TAC were in agreement with biodistribution results of brain tissue. The uptake of [18F]FECNT-d4 in the striatum remained a relatively stable high-plateau period at 5–20 min, whereas the uptake of [18F]FECNT in the striatum began to decrease from 4 min after injection (Fig. 5b). While co-injected with the wellcharacterized specific DAT ligand, the uptake of [18F]FECNT-d4 was mostly inhibited in the target, resulting in the target to non-target ratio close to unity (ST/CB = 1.04). The BPND values of both radioligands were 1.95 ± 0.22 for [18F]FECNT-d4 and 1.65 ± 0.51 for [18F]FECNT. The results indicated that the binding potential of [18F]FECNT-d4 in the rat striatum was higher and more stable than that of [18F]FECNT. The binding potential of [18F]FECNT-d4 in rat striatum was comparable to that of another DAT imaging agent, [18F]FP-CMT (2.50 ± 0.25) [7]. A radiopharmaceutical of another tropane analogue, [18F]LBT999, has a significantly higher BPND (4.39 ± 0.31) in rats [33] and investigated in patients with dopaminergic impairment [34, 35]. However, the effects of metabolites on BPND during prolonged scanning (4 h) are appreciable [36]. Although the binding potential was lower than some of other DAT tracers, [18F]FECNT-d4 still showed good brain uptake and target to non-target ratio, allowing good visualization of DAT in striatal and extra-striatal regions.
After in vitro and in vivo biological studies, we analyzed the metabolism of [18F]FECNT-d4 in vivo. A competent radiotracer should be stable in vivo to retain its biological activity. Similar to cocaine [37], the tropane derivatives would undergo oxidative cleavage of the N-substituent and other metabolic pathways in vivo. For tropane analogues, CYP3A4 exhibited the highest catalytic activity for Ndealkylation [38]. Indeed, in vivo stability studies in rats showed that [18F]FECNT-d4 also transformed to polar metabolite. The major metabolite was issued from Ndealkylation, concomitantly with the departure of the fluoroalkyl chain (formation of fluorine ethanol or its oxidation product). Thirty minutes after the injection, around 69 % of the parent [18F]FECNT-d4 in plasma was transformed, and such polar metabolites of [18F]FECNT have been demonstrated in human and non-human primate plasma [12]. Nevertheless, the metabolic results showed that the parent fraction of [18F]FECNT-d4 in striatum (91 %) was predominant up to 60 min post injection. At 30 min, there were only 16 % of the unchanged [18F]FECNT in plasma and 82 % in striatum. The metabolic stability of [18F]FECNT-d4 observed in rats was superior to that of [18F]FECNT. Interestingly, we also found a more in vivo stability of the tracers in mice than in rats, probably due to the different kinetics of drug metabolism between the species.
Some other 18F-labelled tropane analogs targeting DAT, such as [18F]FP-CMT, [18F]FE-PE2I and [18F]LBP999, have been reported and their in vivo metabolism have been investigated. Metabolite analysis of [18F]FP-CMT produced only one hydrophilic polar metabolite in vivo [7]. The longer carbon chain at the N-substituent seems to produce less oxidative dealkylation in vivo [39]. However, the metabolic analysis in rats displayed that [18F]FP-CMT has a fast metabolic rate with ~88 % parent molecule transformed at 30 min post injection [7].The polar metabolite was supposed to be a 4-carboxy analogue of [18F]FP-CMT, but the metabolite’s brain penetration ability was not be ascertained. Besides Ndealkylation, [18F]FE-PE2I also suffers from hydroxylation and benzylic hydroxylation to yield a series of brainpenetrating metabolites [40]. The in vivo metabolism rate of [18F]FE-PE2I appears rapid kinetics in humans, with 86 % of the parent molecule transformed at 30 min post injection [41]. The prominent accumulation of major radiometabolite 4hydroxymethyl analogue in the striatum suggests a high affinity for DAT as well as 4-carboxy analogue presents nonspecific binding in the brain [41]. These results might confound the quantitative analysis. The radiopharmaceutical of the same chemical class, [18F]LBT999, appears more stable with a slow transformation rate in vivo. There was 60 % unmetabolized plasmatic fraction in humans at 30 min post injection [35], which seems to facilitate clinical imaging. However, the ligand [18F]LBT999 has the same behavior as the biotransformation of PE2I, suggesting that its metabolites also affect PET images and subsequent quantification [42]. Furthermore, the high bone accumulation of the radioactivity implicated high defluorination of [18F]LBT999 [15]. In recent years, in order to overcome these limitations, great efforts have been made to search for better DAT imaging agents. Although the tropane derivative still undergoes metabolism after deuterium substitution, the present study showed that replacing the N-fluoroethyl group of [18F]FECNT with an N-fluoroethyl-d4 could reduce the oxidative cleavage of the N-substituent and led to a significant improvement in the pharmacological profile of this ligand.
Conclusion
In summary, we prepared and characterized a novel deuterated compound [18F]FECNT-d4 based on the tropane structure which was confirmed as a good candidate for DAT imaging. As demonstrated by microPET imaging, in conjunction with biodistribution assays and plasma metabolism studies, [18F]FECNT-d4 displayed a combination of superior brain uptake and better in vivo stability in rats. These results indicate that the 18F-labelled FECNT-d4 tracer is applicable for neuroimaging and worthy of further clinical investigations.
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