Nuclear Medicine and Biology 42 (2015) 226–233

Contents lists available at ScienceDirect

Nuclear Medicine and Biology journal homepage: www.elsevier.com/locate/nucmedbio

Investigation of the chick embryo as a potential alternative to the mouse for evaluation of radiopharmaceuticals Stephanie Haller a, Simon M. Ametamey b, Roger Schibli a,b, Cristina Müller a,⁎ a b

Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul Scherrer Institute, Villigen-PSI, Switzerland Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland

a r t i c l e

i n f o

Article history: Received 4 October 2014 Received in revised form 14 October 2014 Accepted 16 October 2014 Keywords: PET SPECT Chick embryo Radiopharmaceutical Defluorination In vivo stability

a b s t r a c t Introduction: The chick embryo is an emerging in vivo model in several areas of pre-clinical research including radiopharmaceutical sciences. Herein, it was evaluated as a potential test system for assessing the biodistribution and in vivo stability of radiopharmaceuticals. For this purpose, a number of radiopharmaceuticals labeled with 18 125 99m F, I, Tc, and 177Lu were investigated in the chick embryo and compared with the data obtained in mice. Methods: Chick embryos were cultivated ex ovo for 17–19 days before application of the radiopharmaceutical directly into the peritoneum or intravenously using a vein of the chorioallantoic membrane (CAM). At a defined time point after application of radioactivity, the embryos were euthanized by shock-freezing using liquid nitrogen. Afterwards they were separated from residual egg components for post mortem imaging purposes using positron emission tomography (PET) or single photon emission computed tomography (SPECT). Results: SPECT images revealed uptake of [99mTc]pertechnetate and [125I]iodide in the thyroid of chick embryos and mice, whereas [177Lu]lutetium, [18F]fluoride and [99mTc]-methylene diphosphonate ([99mTc]-MDP) were accumulated in the bones. [99mTc]-dimercaptosuccinic acid (99mTc-DMSA) and the somatostatin analog [177Lu]-DOTATOC, as well as the folic acid derivative [ 177Lu]-DOTA-folate showed accumulation in the renal tissue whereas [ 99mTc]-mebrofenin accumulated in the gall bladder and intestine of both species. In vivo dehalogenation of [18F]fallypride and of the folic acid derivative [125I]iodo-tyrosine-folate was observed in both species. In contrast, the 3′-aza-2′-[18F]fluorofolic acid ([18F]-AzaFol) was stable in the chick embryo as well as in the mouse. Conclusions: Our results revealed the same tissue distribution profile and in vivo stability of radiopharmaceuticals in the chick embryo and the mouse. This observation is promising with regard to a potential use of the chick embryo as an inexpensive and simple test model for preclinical screening of novel radiopharmaceuticals. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Replacement of rodents by an evolutionary lower species such as the chick and refinement of animal experiments by using an embryonic development stage which is less sensitive than an adult animal address the principle of the 3Rs, referring to the Replacement, Reduction and Refinement of animal experimentation [1]. The chick embryo and its chorioallantoic membrane (CAM) are widely used in various research fields since this model represents a relatively simple, rapid and inexpensive in vivo test system [2–4]. Over decades, the CAM has been employed for studying angiogenesis as well as tumor growth, its metastasizing potential and cardiovascular development [5]. Recently, the chick embryo and its CAM have also been used in the field of radiopharmaceutical sciences. Würbach et al. described the chick embryo for studying the distribution, uptake and kinetics of [18F] fluoride by using dynamic positron emission tomography (PET) [6]. ⁎ Corresponding author at: Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul Scherrer Institute, CH-5232 Villigen-PSI, Switzerland. Tel.: +41 56 310 44 54; fax: +41 56 310 28 49. E-mail address: [email protected] (C. Müller). http://dx.doi.org/10.1016/j.nucmedbio.2014.10.010 0969-8051/© 2014 Elsevier Inc. All rights reserved.

For this purpose a method was developed that allowed motionartifact-free PET imaging of the chick embryo after the injection of [ 18F]fluoride for visualization of bone uptake. The results in the chick embryo were comparable with data obtained in rodents. Imaging the development of tumors grown on the CAM and angiogenesis could be interesting and a unique possibility for investigation with the chick embryo model [6]. More recently, Warnock et al. demonstrated that glucose metabolism and protein synthesis of glioblastoma tumors grown on the CAM can be visualized by PET imaging after the injection of [18F]fluoro-deoxy-glucose or 2-[ 18F]fluoro-L-tyrosine [7]. The studies point out to the possibility of using the chick embryo as an alternative to the rodent model for the validation of novel PET tracers. The results of these studies were promising and warrant more extensive testing of the chick embryo as an alternative model for testing the in vivo behavior of radiopharmaceuticals [6–8]. It would be convenient to employ a simple technique for initial screening of novel radiopharmaceuticals, especially for investigating the in vivo stability of novel radiotracers at an early development stage. This would allow promoting only the most promising radiotracers to in vivo studies with expensive rodent models while those that fail in the chick embryo would be withdrawn from further testing.

S. Haller et al. / Nuclear Medicine and Biology 42 (2015) 226–233

Novel radiopharmaceutical candidates are first tested by in vitro incubation with blood plasma, specific enzymes or in the case of radiometalated compounds they are usually challenged with chelating agents to investigate potential trans-chelation [9–11]. It is not uncommon to observe high in vitro stability of radiopharmaceuticals if they are tested by these methods [10,12–14]. However, the same radiopharmaceuticals, in particular radiofluorinated and radioiodinated compounds as well as peptide-based radioconjugates, can be decomposed or metabolized immediately after in vivo application [10,13–15]. Therefore, conventional in vitro test systems are of limited value for the prediction of the in vivo behavior of radiopharmaceuticals and, hence, alternative methods are urgently needed. The goal of this work was to explore the feasibility of using the chick embryo as an in vivo model for evaluating the biodistribution and stability of radiopharmaceuticals (Fig. 1). For this purpose we made use of nuclear imaging methods including small-animal PET and single photon emission computed tomography (SPECT). In a first step, we investigated and compared the tissue distribution of various well known and previously reported radiopharmaceuticals in chick embryos and mice. In a second step, the in vivo stability of radioiodinated and radiofluorinated tracers was investigated with regard to potential dehalogenation. The results obtained in the chick embryo were compared with those observed in mice. Finally, the potential of using the chick embryo as a new in vivo test system for future analysis of radiopharmaceuticals is discussed.

227

No-carrier added 177LuCl3 (in HCl 0.04 M, 2 GBq/200 μl) was obtained from Isotope Technologies Garching (ITG GmbH, Garching, Germany). 2.2. Commercial imaging agents and radiopharmaceuticals Dimercaptosuccinic acid (DMSA; TechneScan®, Mallinckrodt, Petten, The Netherlands), methylene diphosphonate (MDP; ROTOP-MDP®, ROTOP Pharmaka AG, Dresden, Germany) and mebrofenin (Bridatec®, GE Healthcare, Braunschweig, Germany) were radiolabeled with [99mTc]technetium according to the instructions of the manufacturers. The somatostatin analog DOTA-[Tyr 3]-octreotide (DOTATOC) was obtained from Advanced Biochemical Compounds GmbH (ABX, Radeberg, Germany). The DOTA-folate conjugate (cm09) was previously developed in our group [16]. Both ligands were radiolabeled with 177Lu at a specific activity of 20 MBq/nmol as previously reported [16]. Sodium diethylene triaminepentaacetic acid (Na-DTPA, 5 mM, pH 5.0, 10 μl) was added for complexation of potential traces of free 177Lu(III) [17]. Quality control was conducted by high performance liquid chromatography (HPLC) as previously reported [16]. [ 125 I]iodo-tyrosine-folate (0.7 MBq/nmol) and 3′-aza-2′-[ 18 F] fluorofolic acid ([ 18F]-AzaFol, 110 GBq/μmol) were prepared as previously reported [9,10]. [ 18F]Fallypride (60 GBq/μmol) was prepared in analogy to a previously reported method on an automated synthesis module by using the tosyl-fallypride precursor (ABX GmbH, Radeberg, Germany) [18]. 2.3. Cultivation of chick embryos

2. Materials and methods 2.1. Radionuclides [ 99mTc]NaTcO4 was eluted from a 99Mo/ 99mTc-generator (Mallinckrodt, Petten, The Netherlands). [ 125I]NaI (in NaOH 10 −5 M, pH 8–11) was purchased from Perkin-Elmer (Waltham, MA, U.S.). Nocarrier added [ 18F]NaF was produced on a 18/9 cyclotron (Ion Beam Applications S.A., IBA, RadioPharma Solutions, Louvain-la-Neuve, Belgium) via the 18O(p,n) 18F-nuclear reaction at the ETH Zurich [9].

The experiments were approved by the local veterinarian department and conducted in accordance with the Swiss law of animal protection. Fertilized chicken eggs were purchased from Animalco AG (Staufen, Switzerland). The eggs were incubated for 72 h at 37 °C with 60% relative humidity. During embryonal development days (EDDs) 1–4, the eggs were rotated every 12 h to prevent accretion of the yolk sack with the egg shell. For the ex ovo cultivation the eggs were cracked at EDD 4 and the embryo including the egg yolk and white was transferred into a sterile plastic bowl and covered with a petri dish. The cultivation was

Fig. 1. Concept of the chick embryo assay for the evaluation of radiopharmaceuticals using PET and SPECT: Fertilized chicken eggs are incubated for 4 days at 37 °C. Then the egg shell is cracked for ex ovo cultivation of the chick embryo. Two weeks later the radiopharmaceutical of interest is administered. After a certain time period the chick embryo is euthanized in liquid nitrogen followed by post mortem imaging.

228

S. Haller et al. / Nuclear Medicine and Biology 42 (2015) 226–233

continued until EDD 17–19 at 37 °C and a relative humidity of 90–100%. Ex ovo cultivation was chosen because it allows better observation and manipulation of the chick embryos. 2.4. Imaging studies in chick embryos PET and SPECT imaging studies with chick embryos were performed between EDD 17 and 19. For injection, the radionuclides and radiopharmaceuticals were diluted in sodium chloride 0.9% or phosphate buffered saline (PBS, pH 7.4) to the required activity concentration. The injection solution of unreacted 177Lu(III) was prepared by adjusting the pH to 4.5–5 with sodium acetate (0.5 M). All compounds were administered intravenously (i.v.) into a vein of the CAM or intraperitoneally (i.p.) into living chick embryos at a volume of 50–100 μl (Table 1). For the studies with [ 125I]iodo-tyrosine-folate, one of the chick embryos was injected with a mixture of the radioconjugate and 4 mg potassium iodide dissolved in 50 μl PBS to block the uptake of potentially released 125 − I into the thyroid glands. Immediately after injection of radioactivity the bowls with the chick embryos were placed back in the incubator at 37 °C. For imaging purposes the chick embryos were euthanized by shock-freezing in liquid nitrogen at different time points after administration of the radioactivity (Table 1). For imaging purposes the embryo was unfrozen in order to separate it from the various egg components in which it was covered. 2.5. Imaging studies in mice The experiments were approved by the local veterinarian department and conducted in accordance with the Swiss law of animal protection. Mice (CD-1 Foxn1nu mice) were obtained at an age of 5–8 weeks from Charles River Laboratories (Sulzfeld, Germany). The radiopharmaceuticals were i.v. injected into the tail vein at a volume of 100 μl. PET and SPECT scans were performed at different time points after administration of the radiopharmaceuticals (Table 1). For in vivo scans, mice were anesthetized with a mixture of isoflurane (1.5–2%) and oxygen. 2.6. PET and SPECT/CT imaging PET scans were performed with a small bench-top preclinical PET scanner (Genisys4, Sofie Biosciences, Culver City, California, U.S.). The energy window ranged from 150 keV to 650 keV and the spatial resolution of reconstructed images was 1.4 mm in full width at half maximum in the center of the field of view (FOV) [19]. Static whole-body PET images lasted between 10 min and 30 min. The data were corrected for random coincidences, decay and dead time. The images were acquired using Genisys4 acquisition software (version 1.2.9.3) and reconstructed with maximum-likelihood expectation maximization (MLEM). Gauss post-reconstruction filtering was performed using the VivoQuant post-processing software (version 1.23, inviCRO Imaging Services and Software, Boston, U.S.).

SPECT and computed tomography (CT) scans were conducted with a four-headed multiplexing multipinhole camera (NanoSPECT/CT, Mediso Medical Imaging Systems, Budapest, Hungary). Each head was fitted with a tungsten collimator of nine pinholes of 1.4 mm. The SPECT/CT images were acquired using Nucline Software (version 1.02; Bioscan Inc., Poway, California, U.S.). The acquisition time per view was chosen depending on the amount of radioactivity at scan start resulting in scan times between 5 min and 4.5 h. CT scans were performed by the integrated CT using a tube voltage of 55 kVp and an exposure time of 1000 ms per view. After acquisition, SPECT data were iteratively reconstructed with HiSPECT software (version 1.4.3049; Scivis GmbH, Göttingen, Germany) using γ-energies of 140.5 keV ± 10% for [ 99mTc]technetium, 28.4 keV ± 10% for [125I]iodine, and 56.1 keV ± 10%, 112.9 keV ± 10% and 208.4 keV ± 10% for [ 177Lu]lutetium. The fused SPECT and CT datasets were post-processed as described for the PET images. 3. Results In this study we investigated several radiopharmaceuticals with regard to their tissue distribution profile and in vivo stability in the chick embryo and the mouse. In order to identify an optimal signal-tobackground ratio SPECT and PET scans were performed at variable time points after injection of the radioactivity. In general, the kinetics was slower in the chick embryo than in the mouse. Therefore several embryos were investigated at variable time points after injection of relatively high amounts of activity in order to determine the most suitable scan time. An image is shown for each radiopharmaceutical that represents the distribution of radioactivity in both the chick embryo and the mouse at the most suitable time point after injection of a particular radiopharmaceutical. 3.1. Accumulation of [125I]iodide and [99mTc]pertechnetate in the thyroid glands SPECT/CT images of the chick embryo which were acquired 1.5 h after the application of 125I − and 3 h after the injection of 99mTcO4− showed accumulation of radioactivity only in the thyroid gland (Fig. 2). At exactly the same site uptake of radioactivity was observed in the mouse 1 h after injection of 125I− and 1.5 h after the application of 99mTcO4− (Fig. 2). 3.2. Imaging bone metabolism with [177Lu]lutetium, [ 18F]fluoride and [99mTc]-MDP After the injection of 177Lu 3+ radioactivity was primarily found in the bones including backbone, beak and limbs of the chick embryo (Fig. 3A). In the mouse the administration of 177Lu 3+ resulted also in uptake of radioactivity in the skeleton and to a lower extent in the kidneys (Fig. 3A). Similar to these findings we also observed

Table 1 Experimental setting of the PET and SPECT studies performed with chick embryos and mice to investigate uptake and potential (in)stability of radiopharmaceuticals. Numbers in parentheses refer to the experiments with mice. Investigation

Compound

Method

Injected radioactivity [MBq]

Scan start [h p.i.]

Scan duration [min]

Fig.

Thyroid gland

TcO4− 125 − I 177 Lu3+ 18 − F [99mTc]-MDP [99mTc]-DMSA [177Lu]-DOTATOC [177Lu]-DOTA-folate [99mTc]-mebrofenin [125I]Iodo-tyrosine-folate [18F]Fallypride [18F]-AzaFol

SPECT SPECT SPECT PET SPECT SPECT SPECT SPECT SPECT SPECT PET PET

20 (50) 5 (6) 15 (34) 10 (1) 50 (60) 60 (45) 30 (36) 10 (34) 22 (30) 1.7 (5) 10 (1.4) 10 (1)

1.5 (1.5) 3 (1) 17 (4) 1 (0.5) 5 (3) 4.5 (3) 5 (5.5) 21 (24) 4/6.5 (0.5) 3 (2) 1.5 (0.8) 1.5 (0.5)

50 (15) 60 (60) 240 (45) 30 (30) 15 (90) 5 (10) 115 (280) 50 (40) 50/15 (5) 20 (40) 10 (10) 10 (10)

2A 2B 3A 3B 3C 4A 4B 4C 5 6A 6B 6C

Bones

Kidneys

Liver Dehalogenation

99m

S. Haller et al. / Nuclear Medicine and Biology 42 (2015) 226–233

229

Fig. 2. SPECT/CT images of chick embryos and mice after application of (A) 99mTcO4− and (B) 125I−. Chick embryos were scanned after the injection of (A) 99mTcO4− (20 MBq; post mortem scan of 50 min) and (B) 125I− (5 MBq; post mortem scan of 60 min). Mice were scanned after injection of (A) 99mTcO4− (50 MBq; in vivo scan of 15 min; anesthesia with isoflurane/oxygen) and (B) 125I− (6 MBq; post mortem scan of 60 min). Uptake of radioactivity was observed in the thyroid gland of both species (yellow arrows).

accumulation of radioactivity in the bones of both species after administration of 18F− and [99mTc]-MDP (Fig. 3B/C).

3.3. Biodistribution of [99mTc]-DMSA, [177Lu]-DOTATOC and [177Lu]-DOTA-folate SPECT/CT studies performed 4.5 h after the administration of [ 99mTc]-DMSA indicated a high kidney uptake of radioactivity in the chick embryo (Fig. 4A). Application of the tumor-targeted radioconjugates [ 177Lu]-DOTATOC and [ 177Lu]-DOTA-folate resulted also in a high renal accumulation of radioactivity in the chick embryos (Fig. 4B/C). In this context it has to be mentioned that the kidneys of the chick embryo are not bean-shaped as it is the case in mice and that they lack a urinary bladder. In contrast, the avian kidneys are three-lobed elongated organs with the ureter ending in the cloaca [20,21]. These results were in agreement with the observation of accumulated radioactivity in the kidneys of mice after the injection of [ 99mTc]-DMSA as well as the [ 177Lu]-radioconjugates (Fig. 4).

3.4. Biodistribution of [99mTc]-mebrofenin [ 99mTc]-mebrofenin resulted in SPECT/CT images that showed accumulation of radioactivity solely in the gall bladder of the chick embryo 4 h after application (Fig. 5A). SPECT/CT acquisitions performed after 6.5 h showed an additional uptake of radioactivity in the chick embryo's intestine (Fig. 5B). The same uptake pattern was also observed in mice where [ 99mTc]-mebrofenin accumulated in the gall bladder and intestine already 30 min after application (Fig. 5C).

3.5. Assessment of in vivo dehalogenation of selected radiopharmaceuticals The potential of the chick embryo as a test model for investigating the in vivo stability of halogenated radiotracers was evaluated on the basis of three selected tracers. The two folate-based radiotracers [125I]iodo-tyrosine-folate and [ 18F]-AzaFol were recently developed in our own group [9,10]. [18F]Fallypride is a well-known radiotracer used as a brain imaging agent in the clinics [22]. Application of [ 125I]iodo-tyrosine-folate

Fig. 3. SPECT and PET images of chick embryos and mice after the application of (A) 177Lu3+, (B) 18F− and (C) [99mTc]-MDP. Chick embryos were scanned after injection of (A) 177Lu3+ (15 MBq; post mortem scan of 240 min), (B) 18F− (10 MBq; post mortem scan of 30 min), and (C) [99mTc]-MDP (50 MBq; post mortem scan of 15 min). Mice were scanned after injection of (A) 177Lu3+ (34 MBq; post mortem scan of 45 min), (B) 18F− (1 MBq; in vivo scan of 30 min; anesthesia with isoflurane/oxygen) and (C) [99mTc]-MDP (60 MBq; post mortem scan of 90 min). Uptake of radioactivity was primarily found in the bones of both species.

230

S. Haller et al. / Nuclear Medicine and Biology 42 (2015) 226–233

Fig. 4. SPECT/CT images of chick embryos and mice after application of (A) [99mTc]-DMSA, (B) [177Lu]-DOTATOC and (C) [177Lu]-DOTA-folate. Chick embryos were scanned after injection of (A) [99mTc]-DMSA (60 MBq; post mortem scan of 5 min), (C) [177Lu]-DOTATOC (30 MBq; post mortem scan of 115 min) and (C) [177Lu]-DOTA-folate (10 MBq; post mortem scan of 50 min). Mice were scanned after injection of (A) 99mTc-DMSA (45 MBq; in vivo scan of 10 min; anesthesia with isoflurane/oxygen), (B) [177Lu]-DOTATOC (36 MBq; post mortem scan of 280 min) and (C) [177Lu]-DOTA-folate (34 MBq; post mortem scan of 40 min). All three radiopharmaceuticals accumulated in the kidneys of both species (white arrows).

resulted in high accumulation of radioactivity in the thyroid glands and gall bladder of the chick embryo 3 h after injection (Fig. 6A) [10]. The uptake in the thyroid glands was blocked if potassium iodide was co-injected (data not shown). The same observation was also made with mice injected with [ 125 I]iodo-tyrosine-folate as previously reported (Fig. 6A) [10]. After the application of [ 18F]-AzaFol to the chick embryo, uptake of radioactivity was found in the kidneys and in the liver similar to what was observed in the mouse (Fig. 6B). PET scans of mice after the injection of [ 18F]fallypride revealed accumulation of radioactivity in the dopaminergic regions of the brain. In addition, radioactivity was seen in the bones, intestine and gall bladder (Fig. 6C). Potential uptake of [ 18F]fallypride in the brain of the chick embryo could not be determined due to the small size and limited resolution of the PET scans. However, accumulation of radioactivity was found in the chick embryo's bones and intestine as it was the case in mice (Fig. 6C).

4. Discussion In this study, we evaluated the chick embryo as a potential test system for radiopharmaceuticals. For this purpose the results obtained in the chick embryos were compared with those observed in mice. With these investigations we aimed for a comparison of metabolic processes and clearance mechanisms of radiopharmaceuticals in chick embryos and mice. In a first step, we investigated the tissue distribution of various radiopharmaceuticals in these two species by nuclear imaging studies. The presence of the sodium iodide symporter located in the basolateral membrane of thyrocytes leads to accumulation of 125I − and 99mTcO4− in the thyroid glands of mice as shown on our SPECT/CT images and in previously reported studies [23–25]. The same site of accumulated radioactivity was also found in the chick embryo. Based on these observations, it is most likely that the sodium iodide symporter

Fig. 5. SPECT/CT images of chick embryos and mice after application of [99mTc]-mebrofenin. Chick embryos were scanned (A) 4 h after application of [99mTc]-mebrofenin (22 MBq; post mortem scan of 50 min) and (B) 6.5 h after application of [99mTc]-mebrofenin (22 MBq; post mortem scan of 15 min). In chick embryos uptake of radioactivity was observed in the gall bladder (red arrow) and in the intestine (green arrow). (C) Mice were scanned 30 min after injection of [99mTc]-mebrofenin (30 MBq; in vivo scan of 5 min; anesthesia with isoflurane/oxygen).

S. Haller et al. / Nuclear Medicine and Biology 42 (2015) 226–233

231

Fig. 6. SPECT/CT images after the administration of radiohalogenated tracers. SPECT scans of chick embryos and mice were performed after injection of (A) [125I]iodo-tyrosine-folate (1.7 MBq; post mortem scan of 20 min, and 5 MBq; post mortem scan of 40 min). Accumulation of radioactivity was observed in the thyroid glands (yellow arrows) and gall bladder (red arrow). PET scans of chick embryos and mice were performed after the injection of (B) [18F]-AzaFol (10 MBq; post mortem scan of 10 min and 1 MBq; in vivo scan of 10 min). Accumulation of radioactivity was found in the kidneys (white arrows) and the liver (orange arrows). Uptake of the radiofolate in the salivary glands was observed in mice (purple arrows). PET scans of chick embryos and mice were performed after injection of (C) [18F]fallypride (10 MBq; post mortem scan of 10 min and, 1.4 MBq; in vivo scan of 10 min). Accumulation of radioactivity was found in the bones and intestine (green arrows). In the mouse radioactivity was also detected in the gall bladder (red arrow) and the dopaminergic brain regions (blue arrows). For in vivo scans the mice were anesthetized with isoflurane/oxygen.

also exists in the chick embryo. For bone imaging, we found that the bone-seeking tracers 18F- and [99mTc]-MDP accumulated in the skeleton of the chick embryo and the mouse. 177Lu3+ was also entrapped in the skeleton of both species as already reported for humans [17]. The reason is the exchange of the lanthanide 177Lu3+ with calcium ions at the bone surface since hydroxyapatite([Ca10(PO4)6(OH)2]-crystal) represents the major part of the bone substance [26,27]. Bone uptake of 18F − is also based on ion exchange in which 18F− replaces hydroxyl-groups of the hydroxyapatite crystal [26]. Other than that, [99mTc]-diphosphonates bind to bone tissue due to physicochemical adsorption to the hydroxyapatite structure and, hence, they mainly accumulate at the mineralization front of the bones [26]. According to our observations, these mechanisms are the same in mice and chick embryos. In a next step, we investigated the tissue distribution of the renal tracer [ 99mTc]-DMSA in the chick embryo and the mouse. In the clinics, [ 99mTc]-DMSA is used for imaging the morphology and localization of the kidneys and for the diagnosis of various kidney diseases [28,29]. Herein, we showed that [ 99mTc]-DMSA accumulates in the renal tissue of chick embryos. For mice, the same observations were made which confirms what was previously reported in the literature [30]. In both species, renal accumulation of radioactivity was observed after administration of [ 177Lu]-DOTATOC and [ 177Lu]-DOTA-folate. [ 177Lu]-DOTATOC is a somatostatin-based radiopeptide used in the clinics for the treatment of neuroendocrine tumors [31]. It is well known that somatostatin analogs accumulate in the renal tissue of patients which may be

problematic with regard to potential radionephropathy after highdosed radionuclide therapy [32]. The uptake of both, [ 99mTc]-DMSA and [ 177Lu]-DOTATOC, in the kidneys of rodents and humans is based on megalin/cubilin receptor-mediated re-absorption in the proximal tubules of the kidneys [30,33]. It is therefore likely that the same uptake mechanism holds true also in the chick embryo. The renal accumulation of [ 177Lu]-DOTA-folate in mice is based on specific binding of radiofolates to the folate receptor which is expressed in the renal proximal tubule cells. This phenomenon has been discussed extensively in the literature [16,34,35]. Most probably the same or a similar mechanism leads to radiofolate uptake in the kidneys of the chick embryo. Based on these hypotheses, the chick embryo could be used for the in vivo evaluation of radioconjugates that are excreted via kidneys and possibly retained due to re-absorption processes. Our SPECT/CT images of chick embryos revealed that [ 99mTc]mebrofenin is released over time from the gall bladder into the intestines. These data are in agreement with what has been reported for mice and humans where a time-dependent clearance of this radiotracer from liver into gall bladder and intestines has been observed [36,37]. The uptake of [ 99mTc]-mebrofenin into human liver is based on various uptake mechanism [38]. The results obtained with [99mTc]-mebrofenin indicate that the hepatic excretion of this radiotracer is comparable between the chick embryo and the mouse. In the second part of our study, we investigated the in vivo stability of the radiohalogenated compounds [125I]iodo-tyrosine-folate, [18F]-AzaFol

232

S. Haller et al. / Nuclear Medicine and Biology 42 (2015) 226–233

Table 2 Advantages and disadvantages of the chick embryo as an in vivo test model for investigation of radiopharmaceuticals. Advantages:

Disadvantages:

Only a simple incubator is required while expensive animal facilities are not needed Daily care of the chick embryos is not necessary

Investigations with the chick embryo are limited to a short and fixed observation period between EDD 17 and EDD19 Little is known about the physiology and metabolism in chick embryos (presence and expression levels of enzymes, transporters, receptors etc.) Cracking the egg shell properly and injecting intravenously are technically challenging interventions which require training of the personnel Analysis of the distribution of radioactivity within organs and tissues such as for instance the brain of the chick embryo is limited by the small size and the resolution of the PET and SPECT scanner

The performance of the in vivo investigation is simple and fast

The use of chick embryos instead of mice is much less expensive (25-fold reduced costs)

The use of the chick embryo instead of mice means a significant contribution to the principle of 3Rs in terms of replacing the adult mouse by a lower species and an embryonic stage which is less sensitive than an adult animal

and [ 18F]fallypride. With regard to in vivo dehalogenation previous studies showed that [ 18F]-AzaFol is stable in rodents whereas [ 125I]iodo-tyrosine-folate and [18F]fallypride are not [9,10,18]. The high accumulation of radioactivity in the thyroid glands of the chick embryo after the application of [ 125I]iodo-tyrosine-folate indicated in vivo deiodination. These results were in agreement with the data obtained for the mouse [10]. Injection of [ 18F]fallypride resulted in accumulation of radioactivity in the bones of the mouse and the chick embryo, indicating in vivo defluorination. In agreement with the results obtained in mice, [ 18F]-AzaFol was stable in the chick embryo. These results showed the same in vivo (in)stability in mice and chick embryos which indicates that metabolic processes are comparable in these species. Based on our observations, the chick embryo is a promising in vivo test model for screening new radiopharmaceuticals. It was observed that sites of accumulation and excretion of various radiopharmaceuticals as well as dehalogenation of radioiodinated and radiofluorinated compounds were comparable in the chick embryo and the mouse. Important advantages of the chick embryo assay are the low costs for growing chick embryos (25-fold reduced compared to the mouse model) and the fact that only a simple lab incubator is needed instead of a cost-intensive animal facility which is necessary for housing mice. Importantly the replacement of rodents by a lower species such as the chick embryo means a significant contribution to the principle of the 3Rs (Replacement, Reduction, Refinement) of animal experiments. In spite of several advantages of using the chick embryo as a test model, there are also a few disadvantages which have to be critically acknowledged (Table 2). The assay is technically challenging in terms of cracking the egg shell and i.v. injection. Besides, the determination and analysis of accumulated radioactivity in small tissues and organs such as the brain are limited by the resolution of the PET or SPECT scanner. Finally, it would be of interest to develop an experimental setting that will allow also dynamic studies instead of only post mortem scans as it was the case in the present study. For this purpose the chick embryo would be cultured in ovo that allows anesthesia of the embryo for in vivo imaging purposes. 5. Conclusion With these studies, we have demonstrated that various radiopharmaceuticals show comparable in vivo behavior in the chick embryo and the mouse. It is therefore likely that the chick embryo could serve as a useful preclinical model for the first evaluation of novel radiopharmaceuticals. More investigations using other radiopharmaceuticals are

warranted to draw final conclusions about the suitability of this model in radiopharmaceutical sciences. Acknowledgments We thank Dr. Thomas Betzel (ETH), Martina Dragic (ETH), Dr. Josefine Reber (PSI), Martin Hungerbühler (PSI) and Dr. Mohammed Farhoud (Sofie Biosciences) for technical assistance. This research was financially supported by the Swiss National Science Foundation (Ambizione-grants PZ00P3_121772 and PZ00P3_138834). References [1] Russell WMS, Burch RL. The principles of humane experimental technique. [Reissued: 1992, Universities Federation for Animal Welfare, Potters Bar, Herts., UK] Special ed. London: Methuen; 1959. [2] Rashidi H, Sottile V. The chick embryo: hatching a model for contemporary biomedical research. BioEssays 2009;31:459–65. [3] Vargas A, Zeisser-Labouebe M, Lange N, Gurny R, Delie F. The chick embryo and its chorioallantoic membrane (CAM) for the in vivo evaluation of drug delivery systems. Adv Drug Deliv Rev 2007;59:1162–76. [4] Lokman NA, Elder AS, Ricciardelli C, Oehler MK. Chick chorioallantoic membrane (CAM) assay as an in vivo model to study the effect of newly identified molecules on ovarian cancer invasion and metastasis. Int J Mol Sci 2012;13:9959–70. [5] Kain KH, Miller JW, Jones-Paris CR, Thomason RT, Lewis JD, Bader DM, et al. The chick embryo as an expanding experimental model for cancer and cardiovascular research. Dev Dyn 2014;243:216–28. [6] Würbach L, Heidrich A, Opfermann T, Gebhardt P, Saluz HP. Insights into bone metabolism of avian embryos in ovo via 3D and 4D 18F-fluoride positron emission tomography. Mol Imaging Biol 2012;14:688–98. [7] Warnock G, Turtoi A, Blomme A, Bretin F, Bahri MA, Lemaire C, et al. In vivo PET/CT in a human glioblastoma chicken chorioallantoic membrane model: a new tool for oncology and radiotracer development. J Nucl Med 2013;54:1782–8. [8] Gebhardt P, Würbach L, Heidrich A, Heinrich L, Walther M, Opfermann T, et al. Dynamic behaviour of selected PET tracers in embryonated chicken eggs. Rev Esp Med Nucl Imagen Mol 2013;32:371–7. [9] Betzel T, Müller C, Groehn V, Muller A, Reber J, Fischer CR, et al. Radiosynthesis and preclinical evaluation of 3′-aza-2′-[18F]fluorofolic acid: a novel PET radiotracer for folate receptor targeting. Bioconjug Chem 2013;24:205–14. [10] Reber J, Struthers H, Betzel T, Hohn A, Schibli R, Müller C. Radioiodinated folic acid conjugates: evaluation of a valuable concept to improve tumor-to-background contrast. Mol Pharm 2012;9:1213–21. [11] Li WP, Ma DS, Higginbotham C, Hoffman T, Ketring AR, Cutler CS, et al. Development of an in vitro model for assessing the in vivo stability of lanthanide chelates. Nucl Med Biol 2001;28:145–54. [12] Müller C, Schubiger PA, Schibli R. In vitro and in vivo targeting of different folate receptor-positive cancer cell lines with a novel 99mTc-radiofolate tracer. Eur J Nucl Med Mol Imaging 2006;33:1162–70. [13] Sephton SM, Dennler P, Leutwiler DS, Mu L, Schibli R, Kramer SD, et al. Development of [18F]-PSS223 as a PET tracer for imaging of metabotropic glutamate receptor subtype 5 (mGluR5). Chimia 2012;66:201–4. [14] Sephton SM, Dennler P, Leutwiler DS, Mu L, Wanger-Baumann CA, Schibli R, et al. Synthesis, radiolabelling and in vitro and in vivo evaluation of a novel fluorinated ABP688 derivative for the PET imaging of metabotropic glutamate receptor subtype 5. Am Nucl Med Mol Imaging 2012;2:14–28. [15] Ocak M, Helbok A, Rangger C, Peitl PK, Nock BA, Morelli G, et al. Comparison of biological stability and metabolism of CCK2 receptor targeting peptides, a collaborative project under COST BM0607. Eur J Nucl Med Mol Imaging 2011;38:1426–35. [16] Müller C, Struthers H, Winiger C, Zhernosekov K, Schibli R. DOTA conjugate with an albumin-binding entity enables the first folic acid-targeted 177Lu-radionuclide tumor therapy in mice. J Nucl Med 2013;54:124–31. [17] Breeman WA, van der Wansem K, Bernard BF, van Gameren A, Erion JL, Visser TJ, et al. The addition of DTPA to [177Lu-DOTA0, Tyr3]-octreotate prior to administration reduces rat skeleton uptake of radioactivity. Eur J Nucl Med Mol Imaging 2003;30: 312–5. [18] Mukherjee J, Yang ZY, Das MK, Brown T. Fluorinated benzamide neuroleptics–III. Development of (S)-N-[(1-allyl-2-pyrrolidinyl)methyl]-5-(3-[18F]fluoropropyl)-2, 3-dimethoxybenzamide as an improved dopamine D-2 receptor tracer. Nucl Med Biol 1995;22:283–96. [19] Herrmann K, Dahlbom M, Nathanson D, Wei L, Radu C, Chatziioannou A, et al. Evaluation of the Genisys4, a bench-top preclinical PET scanner. J Nucl Med 2013; 54:1162–7. [20] Braun EJ. Comparative renal function in reptiles, birds, and mammals. Semin Avian Exot Pet 1998;7:62–71. [21] Bellairs R, Osmond M. The atlas of chick development. 2nd ed. Amsterdam; Boston: Elsevier; 2005. [22] Mukherjee J, Christian BT, Dunigan KA, Shi B, Narayanan TK, Satter M, et al. Brain imaging of 18F-fallypride in normal volunteers: blood analysis, distribution, testretest studies, and preliminary assessment of sensitivity to aging effects on dopamine D-2/D-3 receptors. Synapse 2002;46:170–88. [23] Brandt MP, Kloos RT, Shen DH, Zhang X, Liu YY, Jhiang SM. Micro-single-photon emission computed tomography image acquisition and quantification of sodium-

S. Haller et al. / Nuclear Medicine and Biology 42 (2015) 226–233

[24]

[25]

[26] [27] [28] [29] [30]

[31]

iodide symporter-mediated radionuclide accumulation in mouse thyroid and salivary glands. Thyroid 2012;22:617–24. Franken PR, Guglielmi J, Vanhove C, Koulibaly M, Defrise M, Darcourt J, et al. Distribution and dynamics of 99mTc-pertechnetate uptake in the thyroid and other organs assessed by single-photon emission computed tomography in living mice. Thyroid 2010;20:519–26. Darrouzet E, Lindenthal S, Marcellin D, Pellequer JL, Pourcher T. The sodium/iodide symporter: state of the art of its molecular characterization. Biochim Biophys Acta 1838;2014:244–53. Wong KK, Piert M. Dynamic bone imaging with 99mTc-labeled diphosphonates and 18 F-NaF: mechanisms and applications. J Nucl Med 2013;54:590–9. Müller WA, Linzner U, Schaffer EH. Organ distribution studies of lutetium-177 in mouse. Int J Nucl Med Biol 1978;5:29–31. Durand E, Prigent A. The basics of renal imaging and function studies. Q J Nucl Med 2002;46:249–67. Hjelstuen OK. Technetium-99 m chelators in nuclear medicine. A review. Analyst 1995;120:863–6. Weyer K, Nielsen R, Petersen SV, Christensen EI, Rehling M, Birn H. Renal uptake of 99m Tc-dimercaptosuccinic acid is dependent on normal proximal tubule receptormediated endocytosis. J Nucl Med 2013;54:159–65. van Essen M, Krenning EP, Kam BL, de Jong M, Valkema R, Kwekkeboom DJ. Peptidereceptor radionuclide therapy for endocrine tumors. Nat Rev Endocrinol 2009;5: 382–93.

233

[32] Breeman WA, de Jong M, Kwekkeboom DJ, Valkema R, Bakker WH, Kooij PP, et al. Somatostatin receptor-mediated imaging and therapy: basic science, current knowledge, limitations and future perspectives. Eur J Nucl Med 2001;28:1421–9. [33] Melis M, Krenning EP, Bernard BF, Barone R, Visser TJ, de Jong M. Localisation and mechanism of renal retention of radiolabelled somatostatin analogues. Eur J Nucl Med Mol Imaging 2005;32:1136–43. [34] Holm J, Hansen SI, Hoier-Madsen M, Bostad L. High-affinity folate binding in human choroid plexus. Characterization of radioligand binding, immunoreactivity, molecular heterogeneity and hydrophobic domain of the binding protein. Biochem J 1991;280: 267–71. [35] Parker N, Turk MJ, Westrick E, Lewis JD, Low PS, Leamon CP. Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay. Anal Biochem 2005;338:284–93. [36] Neyt S, Huisman MT, Vanhove C, De Man H, Vliegen M, Moerman L, et al. In vivo visualization and quantification of (Disturbed) Oatp-mediated hepatic uptake and Mrp2-mediated biliary excretion of 99mTc-mebrofenin in mice. J Nucl Med 2013; 54:624–30. [37] Krishnamurthy S, Krishnamurthy GT. Technetium-99m-iminodiacetic acid organic anions: review of biokinetics and clinical application in hepatology. Hepatology 1989;9:139–53. [38] Ghibellini G, Leslie EM, Pollack GM, Brouwer KL. Use of Tc-99m mebrofenin as a clinical probe to assess altered hepatobiliary transport: integration of in vitro, pharmacokinetic modeling, and simulation studies. Pharm Res 2008;25:1851–60.