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Rat Imaging and In Vivo Stability Studies using [11C]-Dimethyl-Diphenyl Ammonium, a Candidate Agent for PET-Myocardial Perfusion Imaging
Nuclear Medicine and Biology 40 (2013) 967–973
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Nuclear Medicine and Biology journal homepage: www.elsevier.com/locate/nucmedbio
Rat Imaging and In Vivo Stability Studies using [ 11C]-Dimethyl-Diphenyl Ammonium, a Candidate Agent for PET-Myocardial Perfusion Imaging☆,☆☆ Orit Jacobson 1, Galith Abourbeh 1, Darya Tsvirkun, Eyal Mishani ⁎ Cyclotron–Radiochemistry–MicroPET Unit, Department of Medical Biophysics and Nuclear Medicine, Hadassah Hebrew University Hospital, Jerusalem 91120, Israel
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Article history: Received 8 May 2013 Received in revised form 7 July 2013 Accepted 9 July 2013 Keywords: Myocardium Perfusion Imaging PET Carbon-11
a b s t r a c t Background: PET myocardial perfusion imaging (MPI) holds several advantages over SPECT for diagnosing coronary artery disease. The short half-lives of prevailing PET-MPI agents hamper wider clinical application of PET in nuclear cardiology; prompting the development of novel PET-MPI agents. We have previously reported on the potential of radiolabeled ammonium salts, and particularly on that of [11C]dimethyl-diphenylammonium ([11C]DMDPA), for cardiac PET imaging. This study was designed to improve the radiosynthesis and increase the yield of [ 11C]DMDPA, characterize more meticulously the kinetics of radioactivity distribution after its injection via micro-PET/CT studies, and further explore its potential for PET-MPI. Methods: The radiosynthetic procedure of [ 11C]DMDPA was improved with respect to the previously reported one. The kinetics of radioactivity distribution following injection of [11C]DMDPA were investigated in juvenile and young adult male SD rats using microPET/CT, and compared to those of [13N]NH3. Furthermore, the metabolic fate of [ 11C]DMDPA in vivo was examined after its injection into rats. Results: Following a radiosynthesis time of 25–27 min, 11.9 ± 1.1 GBq of [ 11C]DMDPA was obtained, with a 43.7% ± 4.3% radiochemical yield (n = 7). Time activity curves calculated after administration of [11C] DMDPA indicated rapid, high and sustained radioactivity uptake in hearts of both juvenile and young adult rats, having a two-fold higher cardiac radioactivity uptake compared to [13N]NH3. Accordingly, at all time points after injection to both juvenile and young adult rats, image quality of the left ventricle was higher with [11C]DMDPA compared to [ 13N]NH3. In vivo stability studies of [11C]DMDPA indicate that no radioactive metabolites could be detected in plasma, liver and urine samples of rats up to 20 min after injection, suggesting that [ 11C]DMDPA is metabolically stable in vivo. Conclusions: This study further illustrates that [ 11C]DMDPA holds, at least in part, essential qualities required from a PET-MPI probe. Owing to the improved radiosynthetic procedure reported herein, [ 11C]DMDPA can be produced in sufficient amounts for clinical use. © 2013 Elsevier Inc. All rights reserved.
1. Introduction The possibility of measuring and quantifying myocardial blood flow (MBF) and coronary flow reserve in absolute terms is a unique and inherent feature of positron emission tomography (PET), provided that a suitable radiopharmaceutical and a proper kinetic model are employed. Moreover, there is increasing evidence indicating that PET offers improved image quality and diagnostic ability compared with single-photon emission computed tomography (SPECT), the dominant methodology in the field of nuclear cardiology [1–6]. These attributes are mostly due to the superior detection ☆ Funding Sources: This work was supported by the Israeli Science Foundation grant number ISF243/11. ☆☆ Disclosures: The authors declare that they have no conflicts of interest. ⁎ Corresponding author. Cyclotron–Radiochemistry–MicroPET Unit, Hadassah Hebrew University Hospital, Jerusalem 91120, Israel. Tel.: +972 2 677 7931; fax: +972 2 641 2033. E-mail address: [email protected] (E. Mishani). 1 These authors equally contributed to the article. 0969-8051/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nucmedbio.2013.07.002
sensitivity and specificity of PET and the advantageous spatial and temporal resolutions of this technique. Employing PET in routine nuclear cardiology imaging could therefore enhance diagnosis and stratification of coronary artery disease (CAD) patients, and those with other related cardiac disorders [1,6–8]. Owing to the establishment of PET as a fundamental diagnostic tool in oncology, the availability of clinical PET and hybrid PET/ computed tomography (CT) cameras is continuously growing. Still, the short half-lives of traditional PET-myocardial perfusion imaging (MPI) agents, such as [ 82Rb], [ 15O]H2O and [ 13N]NH3 are a major impediment to a broader clinical utilization of PET-MPI. Consequently, a growing interest has emerged in recent years in the research and development of novel carbon-11 and fluorine-18labelled potential PET-MPI agents [3,8–15]. Developing new suitable PET-MPI agents, which are labeled with radioisotopes, such as C-11 (t1/2 = 20.3 min) and F-18 (t1/2 = 109.8 min), is a fundamental prerequisite for gaining broader clinical application of PET-MPI. That, together with the recent improvements in PET machinery, in acquisition protocols and in reconstruction algorithms, could help
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establish this cutting-edge technique in routine nuclear cardiology exams, thereby improving diagnostic accuracy and clinical management of CAD patients. Several fluorine-18-labeled radiopharmaceuticals have been investigated as novel potential MPI-agents [3,9–11,15–19], of which, at present, [ 18 F]flurpiridaz is the sole compound known to these authors to have reached advanced clinical trials [8,13,20,21]. Highquality images of the myocardium were demonstrated in different species, including humans, after administration of [ 18 F]flurpiridaz [8,16,17,20,22]. Moreover, the ability to visualize and quantify cardiac defects using this radiopharmaceutical has been well documented in different animal models, as well as in human subjects with perfusion deficits [4,16,21,23]. The availability of a novel PET-cardiac agent with a longer half-life than that of common existing cardiac PET pharmaceuticals could hopefully expand the application of PET in clinical nuclear cardiology, thereby promoting diagnosis and stratification of CAD patients. Quaternary ammonium and phosphonium salts labeled with carbon-11 or fluorine-18 have also been investigated for their potential as PET-MPI agents [9–12,14,15,19]. Of the various carbon11-labeled ammonium salt derivatives developed and studied in our group thus far, [ 11C]-dimethyl-diphenyl-ammonium ([ 11C]DMDPA) has emerged as a favorable PET-MPI candidate [12,14]. High-quality images of the myocardium were obtained in mice, rats and pigs following administration of [ 11C]DMDPA, owing to its high and sustained cardiac uptake and to the high clearance of radioactivity from the lungs and blood pool. Notably, the potential use of [ 11C] DMDPA for visualizing cardiac and/or perfusion defects has been demonstrated in a swine model of permanent partial coronary artery occlusion [14]. This study presents a more detailed analysis of the profile of activity distribution following injection of [ 11C]DMDPA to rats. Specifically, the distribution kinetics of [ 11C]DMDPA in main organs of interest were investigated in juvenile and in young adult rats, and compared with that of [ 13N]NH3 using small animaldedicated PET/CT. In addition, to better characterize the metabolic fate of [ 11C]DMDPA following intravenous (i.v.) administration, its stability in vivo was examined in juvenile and adult rats. Lastly, we report on an improved radiosynthetic procedure and higher radiochemical yields with respect to those previously described [14]; enabling future larger scale studies.
2.2. Chemistry 2.2.1. Radiosynthesis of [ 11C]-Dimethyl-Diphenyl-Ammonium ([ 11C]DMDPA) [ 11C]CO2 (62.5 ± 8.9 GBq (n = 7)) was trapped at − 160 °C. The temperature of the cooling trap was then increased to − 20 °C, and the activity was transferred by a stream of argon (40 mL/min) into reactor 1, containing 300 μL of 0.25 N lithium aluminum hydride (ABX, Radeberg, Germany) in tetrahydrofuran (THF) at − 50 °C. After 2.5 min, the solvent was removed under reduced pressure, and the reactor temperature was increased to 160 °C. Subsequently, hydroiodic acid (Merck) was added, and [ 11C]CH3I was distilled through NaOH (Merck) under argon flow (25 mL/min), followed by a silver triflate column at 200 °C [24]. The ensuing [ 11C]-methyl triflate ([ 11C]CH3OTf) was transferred into a second reactor, containing 20 μL (0.1 mmol) of methyl-diphenyl-amine (Conier, Chongqing, China) in 280 μL dry acetonitrile (Merck) at − 15 °C. At the end of the 1 min distillation step, 27.75 ± 4.44 GBq (n = 7) was trapped in the second reactor, which was sealed and heated to 80 °C for 3 min. Following a 3 min reaction, the solvent was removed under argon flow, at 90 °C. The mixture was cooled to 30 °C, 1.5 mL of acetonitrile/water (1:1) containing 0.1% trifluoroactic acid was added, and the crude product was transferred into a flask containing 8 mL of water and 28 μL NaOH (pH 8). Subsequently, the solution was passed through a Sep-Pak Accell Plus CM light cartridge (Waters, Milford, USA), which was pre-activated with 20 mL HPLC water prior to the synthesis. The cartridge was washed with 12 mL of water, and [ 11C]DMDPA was eluted using 4 mL of sterile isotonic saline (B. Braun, Melsungen, Germany). Identification of the product was determined by a reversed-phase (RP) analytical HPLC system. Quality control analysis was performed on an analytical HPLC, equipped with a variable wavelength UV detector (254 nm) and a radioactivity detector with NaI crystals. A Waters μ-Bondapack C18 column (10 μm, 3.9 mm × 300 mm) was used, with a gradient mobile phase system of acetonitrile: acetate buffer 0.1 M, pH 3.8 (3:7) for 15 min, changing to a solvent ratio of 1:1 for an additional 25 min, at a constant flow rate of 1 mL/min. Identification of [ 11C]DMDPA was confirmed by a co-injection of non-labeled DMDPA, having retention times of 6.87 min and 6.4 min, respectively.
2. Materials and methods
2.3. Biology
2.1. General
2.3.1. MicroPET/CT acquisitions and image analysis Anesthesia was induced, and thereafter maintained by 3.5% and 1.0%–2.5% isoflurane in O2, respectively. All acquisitions were carried out using the Inveon™ multimodality PET-CT small animal-dedicated scanner (Siemens Medical Solutions, USA Inc.). Following a CT scan, PET list-mode acquisition was started at the time of i.v. injection of [ 11C]DMDPA (34 ± 16 MBq, n = 8) or [ 13N] NH3 (77 ± 23 MBq, n = 8). Acquisitions continued for 45 min and 20 min post-injection, for [ 11C]DMDPA and [ 13N]NH3, respectively. Emission sinograms were normalized and corrected for attenuation, scatter, randoms, dead time and decay. Image reconstruction was performed using Fourier rebinning and two dimensional orderedsubsets expectation maximization (2D-OSEM), with a voxel size of 0.776 × 0.776 × 0.796 mm 3. Image analysis and quantification were performed using Inveon Research Workplace 3.1 (Siemens Medical Solutions, USA Inc.). Delineation of volumes of interest (VOIs) was performed by manual segmentation, and the corresponding time activity curves (TACs) were calculated. Distribution of activity was calculated as the percentage of injected dose per mL of tissue (%ID/mL). Standardized uptake values (SUVs) were calculated as the product of %ID/mL of each tissue and the total body weight of the animal.
Unless otherwise stated, chemicals and solvents were purchased from Sigma-Aldrich (Tel Aviv, Israel) and used without further purification. Acetonitrile, HPLC water and ethanol were supplied by Merck (Rosh-Ha'ayin, Israel). Radiosyntheses were carried out on a [ 11C]CH3I module (Nuclear Interface, GE, Munster, Germany). 11C-carbon dioxide was produced by the 14 N(p,α) 11C nuclear reaction on nitrogen containing 0.5% oxygen, using an 18/9 IBA cyclotron. At end of bombardment, the target gas was delivered and trapped by a cryogenic trap in the [ 11C] CH3I module. The synthesis of DMDPA standard was previously published [14]. All animal studies were conducted under a protocol approved by the Animal Research Ethics Committee of the Hebrew University of Jerusalem, and in accordance with its guidelines. Animals were routinely kept in 12 h light/dark cycles and provided with food and water ad libitum. For PET scans, juvenile (~ 4–6 weeks, 114 ± 15 g, n = 8) and young adult (~ 9–11 weeks, 321 ± 35 g, n = 8) male Hsd/SD rats were used. The in vivo stability of [ 11C]DMDPA was investigated in juvenile (126 ± 13 g, n = 4) and young adult Hsd/SD rats (306 ± 11 g, n = 4).
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2.3.2. In vivo stability assay To investigate the in vivo stability, anesthetized (1.0%–2.5% in O2) juvenile and young adult rats were injected i.v with 44 ± 9 MBq/rat (n = 4) or 62 ± 31 MBq/rat (n = 4) of [ 11C]DMDPA respectively. Then, at 10 and 20 min after injection, rats were sacrificed using an intraperitoneal injection of pentobarbitone sodium (CTS chemical industries Ltd., Kiryat Malachi, Israel) (n = 2 per time point). Subsequently, blood and urine were collected and kept over ice; the whole liver was excised, weighed and immersed for 10 min at a ratio of 2:1 (w/v) in cold (4 °C), modified RIPA buffer, containing Tris pH 7.5 (50 mM), NP-40 (EMD Millipore, MA, USA) (1% v/v), sodium deoxycholate (0.5% w/v), sodium pyrophosphate (50 mM), sodium fluoride (100 mM), sodium orthovanadate (5 mM) and NaCl (150 mM) in double distilled water. Then, each liver sample was homogenized using an OMNI TIP™ tissue homogenizer (OMNI Int., Marietta, GA), yielding a homogenous suspension. In the first step of extraction, blood samples were centrifuged (4000 rpm, 5 min) in order to separate the plasma from the red blood cells. Then, radioactivity was extracted from the plasma, urine and liver specimens by adding to each sample an identical volume of a cold acetonitrile: THF (7:3) solution, vortexing for 30 s and centrifuging for 5 min at 10,500 g. Subsequently, the supernatant of each sample was loaded onto Partisil ® LKC18F RP thin layer chromatography (TLC) plates (5 × 20 cm plates; Whatman Int. Ltd., Maidstone, England). TLC plates were run using ethanol/acetate buffer 0.1 M, pH 3.8 (1:1) as mobile phase, and then dried thoroughly. Finally, TLC plates of the extracted samples and another TLC plate onto which a diluted [ 11C] DMDPA solution was loaded as reference, were exposed to a phosphor imager plate (BAS-IP MS 2040 Fuji Photo Film Co., LTD, Japan). In addition, a calibration curve with various concentration of [ 11C]DMDPA was generated, to confirm that the band intensities were within the linear range of the phopshpor imager plate. Phosphor imager plates were scanned with an Image Reader BAS1000 V1.8 scanner for visualization of radioactive bands, and densitometry was carried out using the TINA 2.10 g software. Four replicate samples were prepared from each tissue/organ of each rat. One duplicate was used for loading onto the TLC plates, whereas the other duplicate was treated identically, and measured in a gammacounter (1480 Wizard™ 3″) for calculating the percentage of extraction of radioactivity of each sample. 2.4. Statistical analysis Unless otherwise stated, data are presented as mean ± standard deviation (SD). Group comparisons were made using Student t test for unpaired data. P value was regularly set at 0.05. 3. Results 3.1. [ 11C]DMDPA labeling The two-step, automated radiosynthesis of [ 11C]DMDPA is depicted in Scheme 1. The formation of [ 11C]CH3I was performed in the first reactor, having more than 60% of the activity recovered and transferred to the second reactor, through a silver triflate column, at 200 °C. The extent of conversion of [ 11C]CH3I to [ 11C]CH3OTf varied
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between 50% and 70%. Purification of the crude product was carried out on a Sep-Pak Accell plus CM light cartridge. [ 11C]DMDPA was eluted from the Sep-Pak cartridge using sterile saline, and was further used as is. Overall, following a total radiosynthesis time of 25–27 min, 11.9 ± 1.1 GBq of 11C-DMDPA was obtained (n = 7), with a 43.7% ± 4.3% radiochemical yield, after purification, decay corrected (DC) to the start of synthesis. Radiochemical purity was routinely ≥ 95%, and the specific radioactivity was 28 ± 3 GBq/μmol, decay corrected to end of synthesis (EOS). 3.2. MicroPET imaging Dynamic PET acquisitions were carried out using young adult rats (n = 4), following i.v. administration of [ 11C]DMDPA. The TACs presented in Fig. 1 reveal rapid, high and sustained cardiac uptake, having SUVs of 5.6 and 6.4 at 10 and 45 min after injection, respectively. Furthermore, at all time points after injection, radioactivity levels in the liver were significantly lower than in the heart (p = 0.0003). That, and the rapid clearance of radioactivity from the blood pool and the lungs, resulted in high quality images vis-à-vis heart/tissue radioactivity uptake ratios, as illustrated in Fig. 2. In a comparative study, the kinetics of radioactivity distribution were also investigated following i.v. administration of [ 13N]NH3 into young adult rats (n = 4) (Fig. 1). Compared with [ 11C]DMDPA, at all time points after injection, the cardiac uptake of [ 13N]NH3 was at least 50% lower than that of [ 11C]DMDPA, having SUVs of 2.8 and 2.6 at 10 and 20 min after injection, respectively. Moreover, radioactivity levels in the liver slightly exceeded those of the myocardium, thus yielding lower quality images than those obtained with [ 11C]DMDPA (Fig. 2). In an analogous study, the kinetics of radioactivity distribution were investigated in juvenile SD rats after i.v. injection of either [ 11C] DMDPA (n = 4) or [ 13N]NH3 (n = 4). As illustrated in Fig. 3, cardiac radioactivity uptake values for both [ 11C]DMDPA and [ 13N]NH3 were 40% lower than their respective values in young adult rats. Thus, radioactivity uptake in the myocardium had still remained two-fold higher after administration of [ 11C]DMDPA compared to [ 13N]NH3, using juvenile rats. Interestingly, however, the kinetic of radioactivity distribution in the liver after injection of [ 11C]DMDPA was different in juvenile vs. young adult rats. In the latter, not only were radioactivity levels in the liver consistently lower than those measured in the myocardium, but they were also somewhat constant throughout the acquisition, with SUVs of 4.6 and 4.3 at 10 and 45 min, respectively (Fig. 1). Conversely, radioactivity uptake levels in the liver of juvenile rats had initially exceeded those of the myocardium, reaching a peak SUV of 4.9 at 10 min after injection, and continually decreasing thereafter, to an SUV of 3.2 at 45 min (Fig. 3). Consequently, cardiac radioactivity uptake values following [ 11C]DMDPA injection into juvenile rats were significantly lower than those of the liver throughout the first 25 min of the acquisition (p = 0.03). Thus, improved visualization and quantification of myocardial radioactivity uptake in juvenile rats could be obtained at approximately 35 min after injection of [ 11C]DMDPA. Notably, no such differences in liver distribution kinetics between juvenile and young adult rats were observed following
Scheme 1. The two-step automated radiosynthesis of [11C]DMDPA.
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Fig. 1. TACs obtained following injection of [11C]DMDPA and [13N]NH3 into young adult Hsd/SD rats (295 ± 17 g, n =4 and 348 ± 28 g, n =4, respectively). Results are presented as mean SUV ± SEM.
[ 13N]NH3 administration, wherein liver SUVs had remained roughly constant throughout the acquisition for both groups of rats, with no significant differences in absolute liver radioactivity uptake between the groups. Overall, in both groups of rats, [ 11C]DMDPA demonstrated favorable kinetics for a potential PET-cardiac agent, namely rapid, high and sustained cardiac uptake, combined with high clearance from the lungs and blood pool. Our results in rats indicate that optimal imaging, vis-à-vis myocardial visualization and quantification, could be achieved in both juvenile and young adult rats, yet at different time frames after injection.
the retardation factor (Rf) of [ 11C]DMDPA was 0.51–0.55. TLC radiochromatograms of the extracted radioactivity from different tissues, at several time points after [ 11C]DMDPA injection, are presented in Fig. 5, next to a reference radiochromatogram of a diluted [ 11C]DMDPA standard solution. These results indicate no radioactive metabolites could be detected in plasma, liver and urine samples of young adult rats up to 20 min after injection of [ 11C]DMDPA. The latter samples were also analyzed by analytical radio-HPLC and a radio-TLC scanner, corroborating the results obtained with phosphor imager plates. Identical results were obtained in the group of juvenile rats (data not shown), suggesting that [ 11C]DMDPA is stable in vivo.
3.3. In vivo stability of [ 11C]DMDPA
4. Discussion
The results of the in vivo stability of [ 11C]DMDPA are presented in Fig. 4. Levels of extraction from whole blood were approximately 47% and 45% at 10 and 20 min after injection, respectively, with no significant differences in extraction levels between juvenile and young adult rats. Higher levels of extraction were obtained in plasma and liver samples, wherein about 90% and 85% of the total radioactivity, respectively, were recovered at all investigated time points. Notably, there were no group-differences in total levels of radioactivity extraction from the liver, and the extent of extraction was consistently above 84% for liver samples. The content of extracted radioactivity of each sample was analyzed on RP-TLC, as described in the Methods section. Under these conditions,
Data supporting the power and advantage of PET-MPI are expanding, establishing its strength and utility as an alternative to SPECT in nuclear cardiology. Broader clinical application of PET in this field will be enhanced significantly by the development of wider spectrum of novel, simple and effective PET MPI agents [3,14,15,18,25,26]. Compared to carbon-11 labeling, fluorine-18 labeled pharmaceuticals are generally favored, due to the significantly longer half-life of F-18 and the shorter positron range of the latter, offering a more practical and cost effective use, along with improved image quality. Likewise, C-11 holds similar advantages when compared to nitrogen-13. Previous reports from our group have demonstrated the potential of 11C-labeled ammonium salts for cardiac
Fig. 2. Representative microPET/CT axial (A, D), coronal (B, E) and sagittal (C, F) slice images of radioactivity distribution following injection of [11C]DMDPA (A-C) and [13N]NH3 (D-F) into young adult Hsd/SD rats. Images represent the summation of temporal frames from 2 to 10 min after i.v. injection.
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Fig. 3. TACs obtained following injection of [11C]DMDPA and [13N]NH3 into juvenile Hsd/SD rats (113 ± 8 g, n =4 and 115 ± 21 g, n =4, respectively). Results are presented as mean SUV ± SEM.
PET imaging [12,14]. Structure–activity relationship studies of various 11 C-labeled ammonium salt derivatives were carried out, examining their distribution profiles in mice. From these studies, [ 11C]DMDPA has emerged as a promising candidate for cardiac PET imaging. The present work describes the improvement in the radiosynthesis and purification of [ 11C]DMDPA with respect to the previously published ones [14] and a study in rats which further illustrates that [ 11C] DMDPA holds at least part of the essential traits required from a PETMPI probe. To improve the yield of [ 11C]DMDPA, two major modifications were introduced in the present radiosynthetic procedure, namely (i) using dry acetonitrile instead of methyl ethyl ketone as solvent in the second reactor, and (ii) purification of the final product using cationic solid phase extraction rather than HPLC separation. Prior to the loading of crude material onto the solid phase cartridge, the pH of the crude was adjusted to 8, resulting in the preservation of ≥ 80% of the loaded activity onto the cartridge on the one hand, yet with disposal of the methyl-diphenylamine precursor and of undesired
Fig. 4. Percentage of extracted radioactivity from whole blood and livers of juvenile and young adult SD rats, at different time points after i.v. injection of [11C]DMDPA. Results are presented as mean ± SD (n = 2 per time point per group).
byproducts, on the other. Consequently, under these conditions, the radiosynthesis and purification of [ 11C]DMDPA were significantly upgraded: the overall radiosynthetic procedure time was reduced by ~ 30% (25–27 min vs. 36 min); a three-fold higher radiochemical yield was obtained (~ 44% vs. ~ 13%) and sterile saline was used as vehicle rather than the acidic, 15% ethanol in acetate buffer 0.1 M, which was previously employed [14]. Altogether, following these modifications, [ 11C]DMDPA was obtained at high (≥ 95%) radiochemical purity and at higher activity concentrations (2.9 ± 0.3 GBq/mL, n = 7), sufficient for future larger-scale studies. The distribution kinetics of [ 11C]DMDPA following i.v. administration to young adult SD rats were investigated using microPET/CT imaging, and compared to those of [ 13N]NH3. As presented in Fig. 1, at all time points after injection, the cardiac uptake of [ 11C]DMDPA was at least twice that of [ 13N]NH3, whereas liver radioactivity uptake was approximately 40% higher after injection of [ 11C]DMDPA vs. [ 13N]NH3. Consequently, radioactivity uptake ratios of the heart and adjacent organs like the lungs and liver were consistently higher for [ 11C] DMDPA compared with [ 13N]NH3. Moreover, due to high blood clearance after injection of [ 11C]DMDPA, the radioactivity uptake ratio of heart/blood was consistently greater than 10 in young adult rats. Accordingly, excellent-quality images of the LV could be obtained at early time points after injection of [ 11C]DMDPA (Fig. 2). A similar evaluation of the two radiopharmaceuticals was also carried out in juvenile rats. Comparison of the TACs presented in Figs. 1 and 3 reveals similar radioactivity concentrations in the lungs and blood pool for juvenile and young adult rats following
Fig. 5. TLC radiochromatograms of extracted radioactivity from plasma, liver and urine samples at 10 and 20 min after injection of [11C]DMDPA into young adult SD rats. No radioactive metabolites could be detected up to 20 min after injection, as compared to a diluted [11C]DMDPA standard solution loaded as reference (left).
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administration of both [ 11C]DMDPA and [ 13N]NH3. Contrarily, cardiac radioactivity uptake values had declined to ~ 60% of their corresponding values in young adult rats after administration of either compound. The measured reduction in LV-radioactivity uptake could most likely be attributed to differences in absolute cardiac blood flows between the two groups of rats. Indeed, the higher cardiac uptake values observed in young adult rats following injection of [ 11C] DMDPA and/or [ 13N]NH3 are in excellent agreement with previously reported differences in cardiac flows between juvenile (2.7 mL/min) and young adult (4.7 mL/min) Fischer-344 rats [27]. As previously noted, the kinetics of radioactivity distribution in the liver were different between juvenile and young adult rats following injection of [ 11C]DMDPA, but not after administration of [ 13N]NH3 (Figs. 1 and 3). Being a key metabolic and eliminating organ, analysis and interpretation of radioactivity uptake in the liver are less straightforward. N-13-labeled ammonia is prone to extensive metabolism in both humans [28] and rats [29]. Specifically, intact [ 13N]NH3 represents merely 10% of the total 13N-associated radioactivity measured in rat blood at 5 min after injection [29]. Thus, to better characterize the profile of radioactivity distribution in vivo following injection of [ 11C]DMDPA, its metabolic fate was investigated in juvenile and in young adult rats at 10 and 20 min after injection. To this end, rats were sacrificed, and radioactivity was extracted from whole blood, plasma, liver and urine samples, followed by measurement of each extracted fraction and analysis of its radioactive content using RP-TLC. Blood:plasma partitioning of radioactivity was approximately 1:1, thus only 50% of the blood-associated activity was available for further extraction and analysis (Fig. 4). High extraction levels were measured in liver, plasma and urine samples, having ~ 80%, ~ 90% and 100% of the radioactivity recovered for analysis, respectively. No radioactive metabolites were detected in plasma, liver and urine samples of either young adult (Fig. 5) or juvenile rats (data not shown), suggesting that [ 11C]DMDPA is stable in vivo. Consequently, the differences in liver kinetics between juvenile and young adult rats could not be attributed to the presence of radioactive metabolites. Our unpublished data suggest that the fraction of radioactivity present in the intestine following 11CDMDPA injection is higher in juvenile vs. young adult rats, suggesting that entero-hepatic cycling (EHC) of the parent compound could possibly contribute to the different liver kinetics between the two groups of rats. Being prone to EHC, radioactivity levels in the blood and liver are expected to slightly increase shortly after the initial blood peak associated with the i.v. administration of the compound. Further studies employing arterial blood sampling could help elucidate this hypothesis. TACs of the LV obtained after injection of [ 11C]DMDPA reveal a mild (~ 12%–14%) increase in LV-associated radioactivity from 10 to 45 min after injection, in both juvenile and young adult rats, whereas activity in the LV had remained constant from 10–20 min after injection of [ 13N]NH3 in both groups of rats. A similar rise in LVassociated radioactivity has also been reported for a couple of the foremost investigated radiopharmaceuticals for PET-MPI, namely [ 18 F]fluorobenzyl triphenyl phosphonium ([ 18 F]FBnTP) and [ 18 F] flurpiridaz. In a study published by Sherif and colleagues, an increase of ~ 25% in LV-associated radioactivity has been reported for pigs under rest conditions, but not under adenosine stress [30]. A similar rise (~ 10%) in radioactivity uptake in the heart has also been illustrated for 18 F-FBnTP following its injection to mice [11] and dogs [19] under rest and adenosine stress conditions, respectively. Similar to the LV-TACs obtained for rats in this study, our previously published data for [ 11C]DMDPA studies in pigs indicated a 7%–10% increase in LV-associated radioactivity from 10 to 45 min after injection of [ 11C]DMDPA under rest and stress conditions, whereas no increase in cardiac radioactivity uptake was measured up to 20 min after administration of [ 13 N]NH3 to pigs [14]. Thus, this modest increase in LV-radioactivity uptake with time has been
reported for [ 11C]DMDPA, [ 18 F] flurpiridaz and [ 18 F]FBnTP, yet apparently not for [ 13 N]NH3. The implications of this rise in LVassociated uptake after [ 11C]DMDPA injection vis-à-vis its effects on accuracy of image analysis and quantification of MBF warrant further studies, similar to those reported for [ 18 F]flurpiridaz [30,31]. 5. Conclusions PET-MPI agents hold several advantages over the traditional SPECT pharmaceuticals used for the clinical diagnosis of CAD; prompting the research and development of novel simple and improved PET radiopharmaceuticals. The radiosynthesis and purification of [ 11C] DMDPA have been improved, facilitating future studies in large animals and human subjects. The in vivo rat studies presented herein indicate rapid, elevated and prolonged cardiac uptake of [ 11C]DMDPA, along with high clearance from the lungs and blood pool, resulting in better quality cardiac images compared to those obtained with [ 13N] NH3. Furthermore, under the experimental conditions described above, no radioactive metabolites could be detected after [ 11C] DMDPA injection, suggesting that [ 11C]DMDPA is stable in vivo. Future studies examining the cardiac radioactivity uptake at various rates of myocardial blood flow are warranted to further establish the potential of [ 11C]DMDPA as a PET-MPI agent. Acknowledgments The authors wish to thank Sassi Cohen and Daniel Wajnblum for their invaluable technical support and assistance. References [1] Bengel FM, Higuchi T, Javadi MS, Lautamaki R. Cardiac positron emission tomography. J Am Coll Cardiol 2009;54(1):1–15. [2] Di Carli MF. Cardiac positron emission tomography imaging — state of the art. Asia-Pacific Cardiol 2008;2(1):50–2. [3] Lin X, Zhang J, Wang X, Tang Z, Zhang X, Lu J. Development of radiolabeled compounds for myocardial perfusion imaging. Curr Pharm Des 2012;18(8): 1041–57. [4] Berman DS, Germano G, Slomka PJ. Improvement in PET myocardial perfusion image quality and quantification with flurpiridaz F 18. J Nucl Cardiol 2012;19(Suppl 1):S38–45. [5] Di Carli MF, Dorbala S. Cardiac PET-CT. J Thorac Imaging 2007;22(1):101–6. [6] Saraste A, Kajander S, Han C, Nesterov SV, Knuuti J. PET: is myocardial flow quantification a clinical reality? J Nucl Cardiol 2012;19(5):1044–59. [7] Heller GV, Calnon D, Dorbala S. Recent advances in cardiac PET and PET/CT myocardial perfusion imaging. J Nucl Cardiol 2009;16(6):962–9. [8] Yu M, Nekolla SG, Schwaiger M, Robinson SP. The next generation of cardiac positron emission tomography imaging agents: discovery of flurpiridaz F-18 for detection of coronary disease. Semin Nucl Med 2011;41(4):305–13. [9] Studenov AR, Berridge MS. Synthesis and properties of 18 F-labeled potential myocardial blood flow tracers. Nucl Med Biol 2001;28(6):683–93. [10] Madar I, Ravert HT, Du Y, et al. Characterization of uptake of the new PET imaging compound 18 F-fluorobenzyl triphenyl phosphonium in dog myocardium. J Nucl Med 2006;47(8):1359–66. [11] Madar I, Ravert H, Nelkin B, et al. Characterization of membrane potentialdependent uptake of the novel PET tracer 18 F-fluorobenzyl triphenylphosphonium cation. Eur J Nucl Med Mol Imaging 2007;34(12):2057–65. [12] Ilovich O, Billauer H, Dotan S, Freedman NM, Bocher M, Mishani E. Novel and simple carbon-11-labeled ammonium salts as PET agents for myocardial perfusion imaging. Mol Imaging Biol 2011;13(1):128–39. [13] Nekolla SG, Saraste A. Novel F-18-labeled PET myocardial perfusion tracers: bench to bedside. Curr Cardiol Rep 2011;13(2):145–50. [14] Ilovich O, Abourbeh G, Bocher M, et al. Structure–activity relationship and preclinical evaluation of carbon-11-labeled ammonium salts as PET-myocardial perfusion imaging agents. Mol Imaging Biol 2012;14(5):625–36. [15] Kim DY, Kim HJ, Yu KH, Min JJ. Synthesis of [(18)F]-labeled (2-(2-fluoroethoxy) ethyl)tris(4-methoxyphenyl)phosphonium cation as a potential agent for positron emission tomography myocardial imaging. Nucl Med Biol 2012;39(7): 1093–8. [16] Sherif HM, Saraste A, Weidl E, et al. Evaluation of a novel (18)F-labeled positronemission tomography perfusion tracer for the assessment of myocardial infarct size in rats. Circulation 2009;2(2):77–84. [17] Yu M, Guaraldi M, Kagan M, et al. Assessment of 18 F-labeled mitochondrial complex I inhibitors as PET myocardial perfusion imaging agents in rats, rabbits, and primates. Eur J Nucl Med Mol Imaging 2009;36(1):63–72.
O. Jacobson et al. / Nuclear Medicine and Biology 40 (2013) 967–973 [18] Shoup TM, Elmaleh DR, Brownell AL, Zhu A, Guerrero JL, Fischman AJ. Evaluation of (4-[18 F]fluorophenyl)triphenylphosphonium ion. A potential myocardial blood flow agent for PET. Mol Imaging Biol 2011;13(3):511–7. [19] Madar I, Ravert H, Dipaula A, Du Y, Dannals RF, Becker L. Assessment of severity of coronary artery stenosis in a canine model using the PET agent 18 F-fluorobenzyl triphenyl phosphonium: comparison with 99mTc-tetrofosmin. J Nucl Med 2007;48(6):1021–30. [20] Maddahi J, Czernin J, Lazewatsky J, et al. Phase I, first-in-human study of BMS747158, a novel 18 F-labeled tracer for myocardial perfusion PET: dosimetry, biodistribution, safety, and imaging characteristics after a single injection at rest. J Nucl Med 2011;52(9):1490–8. [21] Maddahi J. Properties of an ideal PET perfusion tracer: new PET tracer cases and data. J Nucl Cardiol 2012;19(Suppl 1):S30–37. [22] Nekolla SG, Reder S, Saraste A, et al. Evaluation of the novel myocardial perfusion positron-emission tomography tracer 18 F-BMS-747158-02: comparison to 13 Nammonia and validation with microspheres in a pig model. Circulation 2009;119(17):2333–42. [23] Higuchi T, Nekolla SG, Huisman MM, et al. A new 18 F-labeled myocardial PET tracer: myocardial uptake after permanent and transient coronary occlusion in rats. J Nucl Med 2008;49(10):1715–22. [24] Bruce H, Mock GK, Ma MTV. Convenient gas phase bromination of [11C]methane and production of [11C]methyl triflate. Nucl Med Biol 1999;26:467–71.
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[25] Higuchi T, Fukushima K, Rischpler C, et al. Stable delineation of the ischemic area by the PET perfusion tracer 18 F-fluorobenzyl triphenyl phosphonium after transient coronary occlusion. J Nucl Med 2011;52(6):965–9. [26] Rischpler C, Park MJ, Fung GS, Javadi M, Tsui BM, Higuchi T. Advances in PET myocardial perfusion imaging: F-18 labeled tracers. Ann Nucl Med 2012;26(1): 1–6. [27] Delp MD, Evans MV, Duan C. Effects of aging on cardiac output, regional blood flow, and body composition in Fischer-344 rats. J Appl Physiol 1998;85(5): 1813–22. [28] Keiding S, Sorensen M, Munk OL, Bender D. Human (13)N-ammonia PET studies: the importance of measuring (13)N-ammonia metabolites in blood. Metab Brain Dis 2010;25(1):49–56. [29] Croteau E, Benard F, Bentourkia M, Rousseau J, Paquette M, Lecomte R. Quantitative myocardial perfusion and coronary reserve in rats with 13 Nammonia and small animal PET: impact of anesthesia and pharmacologic stress agents. J Nucl Med 2004;45(11):1924–30. [30] Sherif HM, Nekolla SG, Saraste A, et al. Simplified quantification of myocardial flow reserve with flurpiridaz F 18: validation with microspheres in a pig model. J Nucl Med 2011;52(4):617–24. [31] Johnson NP, Gould KL. Simplified quantification of myocardial flow reserve with (18)F-flurpiridaz: validation with microspheres in a pig model. J Nucl Med 2011;52(11):1835 [author reply 1835–1836].
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Nuclear Medicine and Biology journal homepage: www.elsevier.com/locate/nucmedbio
Rat Imaging and In Vivo Stability Studies using [ 11C]-Dimethyl-Diphenyl Ammonium, a Candidate Agent for PET-Myocardial Perfusion Imaging☆,☆☆ Orit Jacobson 1, Galith Abourbeh 1, Darya Tsvirkun, Eyal Mishani ⁎ Cyclotron–Radiochemistry–MicroPET Unit, Department of Medical Biophysics and Nuclear Medicine, Hadassah Hebrew University Hospital, Jerusalem 91120, Israel
a r t i c l e
i n f o
Article history: Received 8 May 2013 Received in revised form 7 July 2013 Accepted 9 July 2013 Keywords: Myocardium Perfusion Imaging PET Carbon-11
a b s t r a c t Background: PET myocardial perfusion imaging (MPI) holds several advantages over SPECT for diagnosing coronary artery disease. The short half-lives of prevailing PET-MPI agents hamper wider clinical application of PET in nuclear cardiology; prompting the development of novel PET-MPI agents. We have previously reported on the potential of radiolabeled ammonium salts, and particularly on that of [11C]dimethyl-diphenylammonium ([11C]DMDPA), for cardiac PET imaging. This study was designed to improve the radiosynthesis and increase the yield of [ 11C]DMDPA, characterize more meticulously the kinetics of radioactivity distribution after its injection via micro-PET/CT studies, and further explore its potential for PET-MPI. Methods: The radiosynthetic procedure of [ 11C]DMDPA was improved with respect to the previously reported one. The kinetics of radioactivity distribution following injection of [11C]DMDPA were investigated in juvenile and young adult male SD rats using microPET/CT, and compared to those of [13N]NH3. Furthermore, the metabolic fate of [ 11C]DMDPA in vivo was examined after its injection into rats. Results: Following a radiosynthesis time of 25–27 min, 11.9 ± 1.1 GBq of [ 11C]DMDPA was obtained, with a 43.7% ± 4.3% radiochemical yield (n = 7). Time activity curves calculated after administration of [11C] DMDPA indicated rapid, high and sustained radioactivity uptake in hearts of both juvenile and young adult rats, having a two-fold higher cardiac radioactivity uptake compared to [13N]NH3. Accordingly, at all time points after injection to both juvenile and young adult rats, image quality of the left ventricle was higher with [11C]DMDPA compared to [ 13N]NH3. In vivo stability studies of [11C]DMDPA indicate that no radioactive metabolites could be detected in plasma, liver and urine samples of rats up to 20 min after injection, suggesting that [ 11C]DMDPA is metabolically stable in vivo. Conclusions: This study further illustrates that [ 11C]DMDPA holds, at least in part, essential qualities required from a PET-MPI probe. Owing to the improved radiosynthetic procedure reported herein, [ 11C]DMDPA can be produced in sufficient amounts for clinical use. © 2013 Elsevier Inc. All rights reserved.
1. Introduction The possibility of measuring and quantifying myocardial blood flow (MBF) and coronary flow reserve in absolute terms is a unique and inherent feature of positron emission tomography (PET), provided that a suitable radiopharmaceutical and a proper kinetic model are employed. Moreover, there is increasing evidence indicating that PET offers improved image quality and diagnostic ability compared with single-photon emission computed tomography (SPECT), the dominant methodology in the field of nuclear cardiology [1–6]. These attributes are mostly due to the superior detection ☆ Funding Sources: This work was supported by the Israeli Science Foundation grant number ISF243/11. ☆☆ Disclosures: The authors declare that they have no conflicts of interest. ⁎ Corresponding author. Cyclotron–Radiochemistry–MicroPET Unit, Hadassah Hebrew University Hospital, Jerusalem 91120, Israel. Tel.: +972 2 677 7931; fax: +972 2 641 2033. E-mail address: [email protected] (E. Mishani). 1 These authors equally contributed to the article. 0969-8051/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nucmedbio.2013.07.002
sensitivity and specificity of PET and the advantageous spatial and temporal resolutions of this technique. Employing PET in routine nuclear cardiology imaging could therefore enhance diagnosis and stratification of coronary artery disease (CAD) patients, and those with other related cardiac disorders [1,6–8]. Owing to the establishment of PET as a fundamental diagnostic tool in oncology, the availability of clinical PET and hybrid PET/ computed tomography (CT) cameras is continuously growing. Still, the short half-lives of traditional PET-myocardial perfusion imaging (MPI) agents, such as [ 82Rb], [ 15O]H2O and [ 13N]NH3 are a major impediment to a broader clinical utilization of PET-MPI. Consequently, a growing interest has emerged in recent years in the research and development of novel carbon-11 and fluorine-18labelled potential PET-MPI agents [3,8–15]. Developing new suitable PET-MPI agents, which are labeled with radioisotopes, such as C-11 (t1/2 = 20.3 min) and F-18 (t1/2 = 109.8 min), is a fundamental prerequisite for gaining broader clinical application of PET-MPI. That, together with the recent improvements in PET machinery, in acquisition protocols and in reconstruction algorithms, could help
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establish this cutting-edge technique in routine nuclear cardiology exams, thereby improving diagnostic accuracy and clinical management of CAD patients. Several fluorine-18-labeled radiopharmaceuticals have been investigated as novel potential MPI-agents [3,9–11,15–19], of which, at present, [ 18 F]flurpiridaz is the sole compound known to these authors to have reached advanced clinical trials [8,13,20,21]. Highquality images of the myocardium were demonstrated in different species, including humans, after administration of [ 18 F]flurpiridaz [8,16,17,20,22]. Moreover, the ability to visualize and quantify cardiac defects using this radiopharmaceutical has been well documented in different animal models, as well as in human subjects with perfusion deficits [4,16,21,23]. The availability of a novel PET-cardiac agent with a longer half-life than that of common existing cardiac PET pharmaceuticals could hopefully expand the application of PET in clinical nuclear cardiology, thereby promoting diagnosis and stratification of CAD patients. Quaternary ammonium and phosphonium salts labeled with carbon-11 or fluorine-18 have also been investigated for their potential as PET-MPI agents [9–12,14,15,19]. Of the various carbon11-labeled ammonium salt derivatives developed and studied in our group thus far, [ 11C]-dimethyl-diphenyl-ammonium ([ 11C]DMDPA) has emerged as a favorable PET-MPI candidate [12,14]. High-quality images of the myocardium were obtained in mice, rats and pigs following administration of [ 11C]DMDPA, owing to its high and sustained cardiac uptake and to the high clearance of radioactivity from the lungs and blood pool. Notably, the potential use of [ 11C] DMDPA for visualizing cardiac and/or perfusion defects has been demonstrated in a swine model of permanent partial coronary artery occlusion [14]. This study presents a more detailed analysis of the profile of activity distribution following injection of [ 11C]DMDPA to rats. Specifically, the distribution kinetics of [ 11C]DMDPA in main organs of interest were investigated in juvenile and in young adult rats, and compared with that of [ 13N]NH3 using small animaldedicated PET/CT. In addition, to better characterize the metabolic fate of [ 11C]DMDPA following intravenous (i.v.) administration, its stability in vivo was examined in juvenile and adult rats. Lastly, we report on an improved radiosynthetic procedure and higher radiochemical yields with respect to those previously described [14]; enabling future larger scale studies.
2.2. Chemistry 2.2.1. Radiosynthesis of [ 11C]-Dimethyl-Diphenyl-Ammonium ([ 11C]DMDPA) [ 11C]CO2 (62.5 ± 8.9 GBq (n = 7)) was trapped at − 160 °C. The temperature of the cooling trap was then increased to − 20 °C, and the activity was transferred by a stream of argon (40 mL/min) into reactor 1, containing 300 μL of 0.25 N lithium aluminum hydride (ABX, Radeberg, Germany) in tetrahydrofuran (THF) at − 50 °C. After 2.5 min, the solvent was removed under reduced pressure, and the reactor temperature was increased to 160 °C. Subsequently, hydroiodic acid (Merck) was added, and [ 11C]CH3I was distilled through NaOH (Merck) under argon flow (25 mL/min), followed by a silver triflate column at 200 °C [24]. The ensuing [ 11C]-methyl triflate ([ 11C]CH3OTf) was transferred into a second reactor, containing 20 μL (0.1 mmol) of methyl-diphenyl-amine (Conier, Chongqing, China) in 280 μL dry acetonitrile (Merck) at − 15 °C. At the end of the 1 min distillation step, 27.75 ± 4.44 GBq (n = 7) was trapped in the second reactor, which was sealed and heated to 80 °C for 3 min. Following a 3 min reaction, the solvent was removed under argon flow, at 90 °C. The mixture was cooled to 30 °C, 1.5 mL of acetonitrile/water (1:1) containing 0.1% trifluoroactic acid was added, and the crude product was transferred into a flask containing 8 mL of water and 28 μL NaOH (pH 8). Subsequently, the solution was passed through a Sep-Pak Accell Plus CM light cartridge (Waters, Milford, USA), which was pre-activated with 20 mL HPLC water prior to the synthesis. The cartridge was washed with 12 mL of water, and [ 11C]DMDPA was eluted using 4 mL of sterile isotonic saline (B. Braun, Melsungen, Germany). Identification of the product was determined by a reversed-phase (RP) analytical HPLC system. Quality control analysis was performed on an analytical HPLC, equipped with a variable wavelength UV detector (254 nm) and a radioactivity detector with NaI crystals. A Waters μ-Bondapack C18 column (10 μm, 3.9 mm × 300 mm) was used, with a gradient mobile phase system of acetonitrile: acetate buffer 0.1 M, pH 3.8 (3:7) for 15 min, changing to a solvent ratio of 1:1 for an additional 25 min, at a constant flow rate of 1 mL/min. Identification of [ 11C]DMDPA was confirmed by a co-injection of non-labeled DMDPA, having retention times of 6.87 min and 6.4 min, respectively.
2. Materials and methods
2.3. Biology
2.1. General
2.3.1. MicroPET/CT acquisitions and image analysis Anesthesia was induced, and thereafter maintained by 3.5% and 1.0%–2.5% isoflurane in O2, respectively. All acquisitions were carried out using the Inveon™ multimodality PET-CT small animal-dedicated scanner (Siemens Medical Solutions, USA Inc.). Following a CT scan, PET list-mode acquisition was started at the time of i.v. injection of [ 11C]DMDPA (34 ± 16 MBq, n = 8) or [ 13N] NH3 (77 ± 23 MBq, n = 8). Acquisitions continued for 45 min and 20 min post-injection, for [ 11C]DMDPA and [ 13N]NH3, respectively. Emission sinograms were normalized and corrected for attenuation, scatter, randoms, dead time and decay. Image reconstruction was performed using Fourier rebinning and two dimensional orderedsubsets expectation maximization (2D-OSEM), with a voxel size of 0.776 × 0.776 × 0.796 mm 3. Image analysis and quantification were performed using Inveon Research Workplace 3.1 (Siemens Medical Solutions, USA Inc.). Delineation of volumes of interest (VOIs) was performed by manual segmentation, and the corresponding time activity curves (TACs) were calculated. Distribution of activity was calculated as the percentage of injected dose per mL of tissue (%ID/mL). Standardized uptake values (SUVs) were calculated as the product of %ID/mL of each tissue and the total body weight of the animal.
Unless otherwise stated, chemicals and solvents were purchased from Sigma-Aldrich (Tel Aviv, Israel) and used without further purification. Acetonitrile, HPLC water and ethanol were supplied by Merck (Rosh-Ha'ayin, Israel). Radiosyntheses were carried out on a [ 11C]CH3I module (Nuclear Interface, GE, Munster, Germany). 11C-carbon dioxide was produced by the 14 N(p,α) 11C nuclear reaction on nitrogen containing 0.5% oxygen, using an 18/9 IBA cyclotron. At end of bombardment, the target gas was delivered and trapped by a cryogenic trap in the [ 11C] CH3I module. The synthesis of DMDPA standard was previously published [14]. All animal studies were conducted under a protocol approved by the Animal Research Ethics Committee of the Hebrew University of Jerusalem, and in accordance with its guidelines. Animals were routinely kept in 12 h light/dark cycles and provided with food and water ad libitum. For PET scans, juvenile (~ 4–6 weeks, 114 ± 15 g, n = 8) and young adult (~ 9–11 weeks, 321 ± 35 g, n = 8) male Hsd/SD rats were used. The in vivo stability of [ 11C]DMDPA was investigated in juvenile (126 ± 13 g, n = 4) and young adult Hsd/SD rats (306 ± 11 g, n = 4).
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2.3.2. In vivo stability assay To investigate the in vivo stability, anesthetized (1.0%–2.5% in O2) juvenile and young adult rats were injected i.v with 44 ± 9 MBq/rat (n = 4) or 62 ± 31 MBq/rat (n = 4) of [ 11C]DMDPA respectively. Then, at 10 and 20 min after injection, rats were sacrificed using an intraperitoneal injection of pentobarbitone sodium (CTS chemical industries Ltd., Kiryat Malachi, Israel) (n = 2 per time point). Subsequently, blood and urine were collected and kept over ice; the whole liver was excised, weighed and immersed for 10 min at a ratio of 2:1 (w/v) in cold (4 °C), modified RIPA buffer, containing Tris pH 7.5 (50 mM), NP-40 (EMD Millipore, MA, USA) (1% v/v), sodium deoxycholate (0.5% w/v), sodium pyrophosphate (50 mM), sodium fluoride (100 mM), sodium orthovanadate (5 mM) and NaCl (150 mM) in double distilled water. Then, each liver sample was homogenized using an OMNI TIP™ tissue homogenizer (OMNI Int., Marietta, GA), yielding a homogenous suspension. In the first step of extraction, blood samples were centrifuged (4000 rpm, 5 min) in order to separate the plasma from the red blood cells. Then, radioactivity was extracted from the plasma, urine and liver specimens by adding to each sample an identical volume of a cold acetonitrile: THF (7:3) solution, vortexing for 30 s and centrifuging for 5 min at 10,500 g. Subsequently, the supernatant of each sample was loaded onto Partisil ® LKC18F RP thin layer chromatography (TLC) plates (5 × 20 cm plates; Whatman Int. Ltd., Maidstone, England). TLC plates were run using ethanol/acetate buffer 0.1 M, pH 3.8 (1:1) as mobile phase, and then dried thoroughly. Finally, TLC plates of the extracted samples and another TLC plate onto which a diluted [ 11C] DMDPA solution was loaded as reference, were exposed to a phosphor imager plate (BAS-IP MS 2040 Fuji Photo Film Co., LTD, Japan). In addition, a calibration curve with various concentration of [ 11C]DMDPA was generated, to confirm that the band intensities were within the linear range of the phopshpor imager plate. Phosphor imager plates were scanned with an Image Reader BAS1000 V1.8 scanner for visualization of radioactive bands, and densitometry was carried out using the TINA 2.10 g software. Four replicate samples were prepared from each tissue/organ of each rat. One duplicate was used for loading onto the TLC plates, whereas the other duplicate was treated identically, and measured in a gammacounter (1480 Wizard™ 3″) for calculating the percentage of extraction of radioactivity of each sample. 2.4. Statistical analysis Unless otherwise stated, data are presented as mean ± standard deviation (SD). Group comparisons were made using Student t test for unpaired data. P value was regularly set at 0.05. 3. Results 3.1. [ 11C]DMDPA labeling The two-step, automated radiosynthesis of [ 11C]DMDPA is depicted in Scheme 1. The formation of [ 11C]CH3I was performed in the first reactor, having more than 60% of the activity recovered and transferred to the second reactor, through a silver triflate column, at 200 °C. The extent of conversion of [ 11C]CH3I to [ 11C]CH3OTf varied
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between 50% and 70%. Purification of the crude product was carried out on a Sep-Pak Accell plus CM light cartridge. [ 11C]DMDPA was eluted from the Sep-Pak cartridge using sterile saline, and was further used as is. Overall, following a total radiosynthesis time of 25–27 min, 11.9 ± 1.1 GBq of 11C-DMDPA was obtained (n = 7), with a 43.7% ± 4.3% radiochemical yield, after purification, decay corrected (DC) to the start of synthesis. Radiochemical purity was routinely ≥ 95%, and the specific radioactivity was 28 ± 3 GBq/μmol, decay corrected to end of synthesis (EOS). 3.2. MicroPET imaging Dynamic PET acquisitions were carried out using young adult rats (n = 4), following i.v. administration of [ 11C]DMDPA. The TACs presented in Fig. 1 reveal rapid, high and sustained cardiac uptake, having SUVs of 5.6 and 6.4 at 10 and 45 min after injection, respectively. Furthermore, at all time points after injection, radioactivity levels in the liver were significantly lower than in the heart (p = 0.0003). That, and the rapid clearance of radioactivity from the blood pool and the lungs, resulted in high quality images vis-à-vis heart/tissue radioactivity uptake ratios, as illustrated in Fig. 2. In a comparative study, the kinetics of radioactivity distribution were also investigated following i.v. administration of [ 13N]NH3 into young adult rats (n = 4) (Fig. 1). Compared with [ 11C]DMDPA, at all time points after injection, the cardiac uptake of [ 13N]NH3 was at least 50% lower than that of [ 11C]DMDPA, having SUVs of 2.8 and 2.6 at 10 and 20 min after injection, respectively. Moreover, radioactivity levels in the liver slightly exceeded those of the myocardium, thus yielding lower quality images than those obtained with [ 11C]DMDPA (Fig. 2). In an analogous study, the kinetics of radioactivity distribution were investigated in juvenile SD rats after i.v. injection of either [ 11C] DMDPA (n = 4) or [ 13N]NH3 (n = 4). As illustrated in Fig. 3, cardiac radioactivity uptake values for both [ 11C]DMDPA and [ 13N]NH3 were 40% lower than their respective values in young adult rats. Thus, radioactivity uptake in the myocardium had still remained two-fold higher after administration of [ 11C]DMDPA compared to [ 13N]NH3, using juvenile rats. Interestingly, however, the kinetic of radioactivity distribution in the liver after injection of [ 11C]DMDPA was different in juvenile vs. young adult rats. In the latter, not only were radioactivity levels in the liver consistently lower than those measured in the myocardium, but they were also somewhat constant throughout the acquisition, with SUVs of 4.6 and 4.3 at 10 and 45 min, respectively (Fig. 1). Conversely, radioactivity uptake levels in the liver of juvenile rats had initially exceeded those of the myocardium, reaching a peak SUV of 4.9 at 10 min after injection, and continually decreasing thereafter, to an SUV of 3.2 at 45 min (Fig. 3). Consequently, cardiac radioactivity uptake values following [ 11C]DMDPA injection into juvenile rats were significantly lower than those of the liver throughout the first 25 min of the acquisition (p = 0.03). Thus, improved visualization and quantification of myocardial radioactivity uptake in juvenile rats could be obtained at approximately 35 min after injection of [ 11C]DMDPA. Notably, no such differences in liver distribution kinetics between juvenile and young adult rats were observed following
Scheme 1. The two-step automated radiosynthesis of [11C]DMDPA.
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Fig. 1. TACs obtained following injection of [11C]DMDPA and [13N]NH3 into young adult Hsd/SD rats (295 ± 17 g, n =4 and 348 ± 28 g, n =4, respectively). Results are presented as mean SUV ± SEM.
[ 13N]NH3 administration, wherein liver SUVs had remained roughly constant throughout the acquisition for both groups of rats, with no significant differences in absolute liver radioactivity uptake between the groups. Overall, in both groups of rats, [ 11C]DMDPA demonstrated favorable kinetics for a potential PET-cardiac agent, namely rapid, high and sustained cardiac uptake, combined with high clearance from the lungs and blood pool. Our results in rats indicate that optimal imaging, vis-à-vis myocardial visualization and quantification, could be achieved in both juvenile and young adult rats, yet at different time frames after injection.
the retardation factor (Rf) of [ 11C]DMDPA was 0.51–0.55. TLC radiochromatograms of the extracted radioactivity from different tissues, at several time points after [ 11C]DMDPA injection, are presented in Fig. 5, next to a reference radiochromatogram of a diluted [ 11C]DMDPA standard solution. These results indicate no radioactive metabolites could be detected in plasma, liver and urine samples of young adult rats up to 20 min after injection of [ 11C]DMDPA. The latter samples were also analyzed by analytical radio-HPLC and a radio-TLC scanner, corroborating the results obtained with phosphor imager plates. Identical results were obtained in the group of juvenile rats (data not shown), suggesting that [ 11C]DMDPA is stable in vivo.
3.3. In vivo stability of [ 11C]DMDPA
4. Discussion
The results of the in vivo stability of [ 11C]DMDPA are presented in Fig. 4. Levels of extraction from whole blood were approximately 47% and 45% at 10 and 20 min after injection, respectively, with no significant differences in extraction levels between juvenile and young adult rats. Higher levels of extraction were obtained in plasma and liver samples, wherein about 90% and 85% of the total radioactivity, respectively, were recovered at all investigated time points. Notably, there were no group-differences in total levels of radioactivity extraction from the liver, and the extent of extraction was consistently above 84% for liver samples. The content of extracted radioactivity of each sample was analyzed on RP-TLC, as described in the Methods section. Under these conditions,
Data supporting the power and advantage of PET-MPI are expanding, establishing its strength and utility as an alternative to SPECT in nuclear cardiology. Broader clinical application of PET in this field will be enhanced significantly by the development of wider spectrum of novel, simple and effective PET MPI agents [3,14,15,18,25,26]. Compared to carbon-11 labeling, fluorine-18 labeled pharmaceuticals are generally favored, due to the significantly longer half-life of F-18 and the shorter positron range of the latter, offering a more practical and cost effective use, along with improved image quality. Likewise, C-11 holds similar advantages when compared to nitrogen-13. Previous reports from our group have demonstrated the potential of 11C-labeled ammonium salts for cardiac
Fig. 2. Representative microPET/CT axial (A, D), coronal (B, E) and sagittal (C, F) slice images of radioactivity distribution following injection of [11C]DMDPA (A-C) and [13N]NH3 (D-F) into young adult Hsd/SD rats. Images represent the summation of temporal frames from 2 to 10 min after i.v. injection.
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Fig. 3. TACs obtained following injection of [11C]DMDPA and [13N]NH3 into juvenile Hsd/SD rats (113 ± 8 g, n =4 and 115 ± 21 g, n =4, respectively). Results are presented as mean SUV ± SEM.
PET imaging [12,14]. Structure–activity relationship studies of various 11 C-labeled ammonium salt derivatives were carried out, examining their distribution profiles in mice. From these studies, [ 11C]DMDPA has emerged as a promising candidate for cardiac PET imaging. The present work describes the improvement in the radiosynthesis and purification of [ 11C]DMDPA with respect to the previously published ones [14] and a study in rats which further illustrates that [ 11C] DMDPA holds at least part of the essential traits required from a PETMPI probe. To improve the yield of [ 11C]DMDPA, two major modifications were introduced in the present radiosynthetic procedure, namely (i) using dry acetonitrile instead of methyl ethyl ketone as solvent in the second reactor, and (ii) purification of the final product using cationic solid phase extraction rather than HPLC separation. Prior to the loading of crude material onto the solid phase cartridge, the pH of the crude was adjusted to 8, resulting in the preservation of ≥ 80% of the loaded activity onto the cartridge on the one hand, yet with disposal of the methyl-diphenylamine precursor and of undesired
Fig. 4. Percentage of extracted radioactivity from whole blood and livers of juvenile and young adult SD rats, at different time points after i.v. injection of [11C]DMDPA. Results are presented as mean ± SD (n = 2 per time point per group).
byproducts, on the other. Consequently, under these conditions, the radiosynthesis and purification of [ 11C]DMDPA were significantly upgraded: the overall radiosynthetic procedure time was reduced by ~ 30% (25–27 min vs. 36 min); a three-fold higher radiochemical yield was obtained (~ 44% vs. ~ 13%) and sterile saline was used as vehicle rather than the acidic, 15% ethanol in acetate buffer 0.1 M, which was previously employed [14]. Altogether, following these modifications, [ 11C]DMDPA was obtained at high (≥ 95%) radiochemical purity and at higher activity concentrations (2.9 ± 0.3 GBq/mL, n = 7), sufficient for future larger-scale studies. The distribution kinetics of [ 11C]DMDPA following i.v. administration to young adult SD rats were investigated using microPET/CT imaging, and compared to those of [ 13N]NH3. As presented in Fig. 1, at all time points after injection, the cardiac uptake of [ 11C]DMDPA was at least twice that of [ 13N]NH3, whereas liver radioactivity uptake was approximately 40% higher after injection of [ 11C]DMDPA vs. [ 13N]NH3. Consequently, radioactivity uptake ratios of the heart and adjacent organs like the lungs and liver were consistently higher for [ 11C] DMDPA compared with [ 13N]NH3. Moreover, due to high blood clearance after injection of [ 11C]DMDPA, the radioactivity uptake ratio of heart/blood was consistently greater than 10 in young adult rats. Accordingly, excellent-quality images of the LV could be obtained at early time points after injection of [ 11C]DMDPA (Fig. 2). A similar evaluation of the two radiopharmaceuticals was also carried out in juvenile rats. Comparison of the TACs presented in Figs. 1 and 3 reveals similar radioactivity concentrations in the lungs and blood pool for juvenile and young adult rats following
Fig. 5. TLC radiochromatograms of extracted radioactivity from plasma, liver and urine samples at 10 and 20 min after injection of [11C]DMDPA into young adult SD rats. No radioactive metabolites could be detected up to 20 min after injection, as compared to a diluted [11C]DMDPA standard solution loaded as reference (left).
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administration of both [ 11C]DMDPA and [ 13N]NH3. Contrarily, cardiac radioactivity uptake values had declined to ~ 60% of their corresponding values in young adult rats after administration of either compound. The measured reduction in LV-radioactivity uptake could most likely be attributed to differences in absolute cardiac blood flows between the two groups of rats. Indeed, the higher cardiac uptake values observed in young adult rats following injection of [ 11C] DMDPA and/or [ 13N]NH3 are in excellent agreement with previously reported differences in cardiac flows between juvenile (2.7 mL/min) and young adult (4.7 mL/min) Fischer-344 rats [27]. As previously noted, the kinetics of radioactivity distribution in the liver were different between juvenile and young adult rats following injection of [ 11C]DMDPA, but not after administration of [ 13N]NH3 (Figs. 1 and 3). Being a key metabolic and eliminating organ, analysis and interpretation of radioactivity uptake in the liver are less straightforward. N-13-labeled ammonia is prone to extensive metabolism in both humans [28] and rats [29]. Specifically, intact [ 13N]NH3 represents merely 10% of the total 13N-associated radioactivity measured in rat blood at 5 min after injection [29]. Thus, to better characterize the profile of radioactivity distribution in vivo following injection of [ 11C]DMDPA, its metabolic fate was investigated in juvenile and in young adult rats at 10 and 20 min after injection. To this end, rats were sacrificed, and radioactivity was extracted from whole blood, plasma, liver and urine samples, followed by measurement of each extracted fraction and analysis of its radioactive content using RP-TLC. Blood:plasma partitioning of radioactivity was approximately 1:1, thus only 50% of the blood-associated activity was available for further extraction and analysis (Fig. 4). High extraction levels were measured in liver, plasma and urine samples, having ~ 80%, ~ 90% and 100% of the radioactivity recovered for analysis, respectively. No radioactive metabolites were detected in plasma, liver and urine samples of either young adult (Fig. 5) or juvenile rats (data not shown), suggesting that [ 11C]DMDPA is stable in vivo. Consequently, the differences in liver kinetics between juvenile and young adult rats could not be attributed to the presence of radioactive metabolites. Our unpublished data suggest that the fraction of radioactivity present in the intestine following 11CDMDPA injection is higher in juvenile vs. young adult rats, suggesting that entero-hepatic cycling (EHC) of the parent compound could possibly contribute to the different liver kinetics between the two groups of rats. Being prone to EHC, radioactivity levels in the blood and liver are expected to slightly increase shortly after the initial blood peak associated with the i.v. administration of the compound. Further studies employing arterial blood sampling could help elucidate this hypothesis. TACs of the LV obtained after injection of [ 11C]DMDPA reveal a mild (~ 12%–14%) increase in LV-associated radioactivity from 10 to 45 min after injection, in both juvenile and young adult rats, whereas activity in the LV had remained constant from 10–20 min after injection of [ 13N]NH3 in both groups of rats. A similar rise in LVassociated radioactivity has also been reported for a couple of the foremost investigated radiopharmaceuticals for PET-MPI, namely [ 18 F]fluorobenzyl triphenyl phosphonium ([ 18 F]FBnTP) and [ 18 F] flurpiridaz. In a study published by Sherif and colleagues, an increase of ~ 25% in LV-associated radioactivity has been reported for pigs under rest conditions, but not under adenosine stress [30]. A similar rise (~ 10%) in radioactivity uptake in the heart has also been illustrated for 18 F-FBnTP following its injection to mice [11] and dogs [19] under rest and adenosine stress conditions, respectively. Similar to the LV-TACs obtained for rats in this study, our previously published data for [ 11C]DMDPA studies in pigs indicated a 7%–10% increase in LV-associated radioactivity from 10 to 45 min after injection of [ 11C]DMDPA under rest and stress conditions, whereas no increase in cardiac radioactivity uptake was measured up to 20 min after administration of [ 13 N]NH3 to pigs [14]. Thus, this modest increase in LV-radioactivity uptake with time has been
reported for [ 11C]DMDPA, [ 18 F] flurpiridaz and [ 18 F]FBnTP, yet apparently not for [ 13 N]NH3. The implications of this rise in LVassociated uptake after [ 11C]DMDPA injection vis-à-vis its effects on accuracy of image analysis and quantification of MBF warrant further studies, similar to those reported for [ 18 F]flurpiridaz [30,31]. 5. Conclusions PET-MPI agents hold several advantages over the traditional SPECT pharmaceuticals used for the clinical diagnosis of CAD; prompting the research and development of novel simple and improved PET radiopharmaceuticals. The radiosynthesis and purification of [ 11C] DMDPA have been improved, facilitating future studies in large animals and human subjects. The in vivo rat studies presented herein indicate rapid, elevated and prolonged cardiac uptake of [ 11C]DMDPA, along with high clearance from the lungs and blood pool, resulting in better quality cardiac images compared to those obtained with [ 13N] NH3. Furthermore, under the experimental conditions described above, no radioactive metabolites could be detected after [ 11C] DMDPA injection, suggesting that [ 11C]DMDPA is stable in vivo. Future studies examining the cardiac radioactivity uptake at various rates of myocardial blood flow are warranted to further establish the potential of [ 11C]DMDPA as a PET-MPI agent. Acknowledgments The authors wish to thank Sassi Cohen and Daniel Wajnblum for their invaluable technical support and assistance. References [1] Bengel FM, Higuchi T, Javadi MS, Lautamaki R. Cardiac positron emission tomography. J Am Coll Cardiol 2009;54(1):1–15. [2] Di Carli MF. Cardiac positron emission tomography imaging — state of the art. Asia-Pacific Cardiol 2008;2(1):50–2. [3] Lin X, Zhang J, Wang X, Tang Z, Zhang X, Lu J. Development of radiolabeled compounds for myocardial perfusion imaging. Curr Pharm Des 2012;18(8): 1041–57. [4] Berman DS, Germano G, Slomka PJ. Improvement in PET myocardial perfusion image quality and quantification with flurpiridaz F 18. J Nucl Cardiol 2012;19(Suppl 1):S38–45. [5] Di Carli MF, Dorbala S. Cardiac PET-CT. J Thorac Imaging 2007;22(1):101–6. [6] Saraste A, Kajander S, Han C, Nesterov SV, Knuuti J. PET: is myocardial flow quantification a clinical reality? J Nucl Cardiol 2012;19(5):1044–59. [7] Heller GV, Calnon D, Dorbala S. Recent advances in cardiac PET and PET/CT myocardial perfusion imaging. J Nucl Cardiol 2009;16(6):962–9. [8] Yu M, Nekolla SG, Schwaiger M, Robinson SP. The next generation of cardiac positron emission tomography imaging agents: discovery of flurpiridaz F-18 for detection of coronary disease. Semin Nucl Med 2011;41(4):305–13. [9] Studenov AR, Berridge MS. Synthesis and properties of 18 F-labeled potential myocardial blood flow tracers. Nucl Med Biol 2001;28(6):683–93. [10] Madar I, Ravert HT, Du Y, et al. Characterization of uptake of the new PET imaging compound 18 F-fluorobenzyl triphenyl phosphonium in dog myocardium. J Nucl Med 2006;47(8):1359–66. [11] Madar I, Ravert H, Nelkin B, et al. Characterization of membrane potentialdependent uptake of the novel PET tracer 18 F-fluorobenzyl triphenylphosphonium cation. Eur J Nucl Med Mol Imaging 2007;34(12):2057–65. [12] Ilovich O, Billauer H, Dotan S, Freedman NM, Bocher M, Mishani E. Novel and simple carbon-11-labeled ammonium salts as PET agents for myocardial perfusion imaging. Mol Imaging Biol 2011;13(1):128–39. [13] Nekolla SG, Saraste A. Novel F-18-labeled PET myocardial perfusion tracers: bench to bedside. Curr Cardiol Rep 2011;13(2):145–50. [14] Ilovich O, Abourbeh G, Bocher M, et al. Structure–activity relationship and preclinical evaluation of carbon-11-labeled ammonium salts as PET-myocardial perfusion imaging agents. Mol Imaging Biol 2012;14(5):625–36. [15] Kim DY, Kim HJ, Yu KH, Min JJ. Synthesis of [(18)F]-labeled (2-(2-fluoroethoxy) ethyl)tris(4-methoxyphenyl)phosphonium cation as a potential agent for positron emission tomography myocardial imaging. Nucl Med Biol 2012;39(7): 1093–8. [16] Sherif HM, Saraste A, Weidl E, et al. Evaluation of a novel (18)F-labeled positronemission tomography perfusion tracer for the assessment of myocardial infarct size in rats. Circulation 2009;2(2):77–84. [17] Yu M, Guaraldi M, Kagan M, et al. Assessment of 18 F-labeled mitochondrial complex I inhibitors as PET myocardial perfusion imaging agents in rats, rabbits, and primates. Eur J Nucl Med Mol Imaging 2009;36(1):63–72.
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