Development of new copper-64 labeled rhodamine: a potential PET myocardial perfusion imaging agent

Background Myocardial perfusion imaging (MPI) is one of the most commonly performed investigations in nuclear medicine procedures. Due to the longer half-life of the emerging positron emitter copper-64 and its availability from low energy cyclotron, together with its well-known coordination chemistry, we have synthesized 64Cu-labeled NOTA- and 64Cu-NOTAM-rhodamine conjugates as potential cardiac imaging agents using PET. Results 64Cu-NOTA- and 64Cu-NOTAM-rhodamine conjugates were synthesized using a traightforward and one-step simple reaction. Radiochemical yields were greater than 97% (decay corrected), with a total synthesis time of less than 25 min. Radiochemical purities were always greater than 98% as assessed by TLC and HPLC. These synthetic approaches hold considerable promise as a simple method for 64Cu-rhodamine conjugates synthesis, with high radiochemical yield and purity. Biodistribution studies in normal Fischer rats at 60 min post-injection, demonstrated significant heart uptake and a good biodistribution profile for both the radioconjugates. However, the 64Cu-NOTAM-rhodamine conjugate has shown more heart uptake (~ 10% ID/g) over the 64Cu-NOTA-rhodamine conjugate (5.6% ID/g). Conclusions These results demonstrate that these radioconjugates may be useful probes for the PET evaluation of MPI.

F-labeled radiopharmaceuticals for MPI have been prepared and evaluated and some of these new agents have shown better image quality and a better association to true myocardial blood flow than 99m Tc-MIBI (Marshall et al. 2004;Madar et al. 2006;Yu et al. 2007;Huisman et al. 2008;Shoup et al. 2011).
It has been shown that the rhodamine dyes are accumulated in mitochondria and take around 30% of the myocardial intracellular volume in the heart (Kronauge et al. 1992). Thus, numerous 18 F-rhodamines analogs as potential MPI agents were developed recently Gottumukkala et al. 2010; Bartholoma et al. 2012). In particular, 18 F-labeled rhodamine B diethylene glycol ester ([ 18 F]RhoBDEGF) has provided an excellent image quality and might be a potential PET tracer for MPI studies (Storey et al. 1993). Recently, our group has developed [ 18 F]-FDG-rhodamine, [ 124 I]-SIB-rhodamine, and 68 Ga-NOTA-rhodamine conjugates. These radioconjugates have demonstrated a high myocardial uptake and favorable pharmacokinetics which indicate that some of these radioconjugates may be useful for MPI studies (Aljammaz et al. 2014(Aljammaz et al. , 2015a(Aljammaz et al. , b, 2019. The cyclotron-produced positron emitter copper-64 ( 64 Cu) together with its 12.7 h half-life and well-known coordination chemistry makes it one of the most attractive radionuclides for PET imaging (McCarthy et al. 1997;Alliot et al. 2011;Szelecsenyi et al. 1993). Therefore, varieties of 64 Cu-radiolabeled biomolecules for potential use beyond the measurement of glucose metabolism were developed and investigated (Anderson and Ferdani 2009;Zhang et al. 2013;Sprague et al. 2007;Hao et al. 2009;Evangelista et al. 2013). Among these, 64 Cu-labeled DOTA-somatostatin conjugate ( 64 Cu-DOTA-TATE) has been recently approved by the FDA for the localization of somatostatin receptor-positive neuroendocrine tumors (NETs) in adult patients. For the past several years, we are interested in developing new agents for MPI studies; in this paper, we described the synthesis and initial evaluation of the 64 Cu-NOTA-and 64 Cu-NOTAMrhodamine conjugates.

Chemistry
The synthetic methods for the preparation of NOTA-and NOTAM-rhodamines are mentioned in Schemes 1 and 2. These conjugates were fully characterized by HPLC and the mass spectral data and agreed with the expected structures. The precursor's NOTA-and NOTAM-rhodamine conjugates were obtained as an off-white Page 3 of 15 AlHokbany et al. EJNMMI Radiopharmacy and Chemistry (2022) 7:19 precipitate in 60% and 20% yield, respectively. The theoretical calculated molecular masses for NOTA-and NOTAM-rhodamine conjugates were 658 and 628, respectively. These values agreed well with the attained ES-MS [M + 1] + = 659 and 629, respectively. Chemical purities for NOTA-and NOTAM-rhodamine conjugates were higher than 98% as assessed by HPLC. The reference Cu II -NOTA-and Cu II -NOTAM-rhodamine conjugates were obtained as off-white powders with overall chemical yields of ~ 50%, which appear to decompose beyond 180 °C. The calculated molecular masses for Cu II -NOTA-and Cu II -NOTAM-rhodamine were 720 and 690, respectively. These values were agreed with the attained ES-MS [M + 1] + = 721 and 691, respectively. Chemical purities of Cu II -NOTA-and Cu II -NOTAM-rhodamine were higher than 97% as assessed by HPLC with retention times of 11.6 and 13.1 min, respectively.

Stability in plasma
The proteolytic degradation of the 64 Cu-NOTA-and 64 Cu-NOTAM-rhodamine was calculated in human plasma in vitro. HPLC analysis of the plasma samples revealed that the 64 Cu-NOTA-and 64 Cu-NOTAM-rhodamine remained highly stable (> 97%) during incubation at 37 °C for at least 4 h, suggesting a high in vitro stability of these radiolabeled bioconjugates.

In vivo Biodistribution
Preliminary biological properties of 64 Cu-NOTA-and 64 Cu-NOTAM-rhodamine in normal Fischer rats at 60 min p.i. are summarized in Table 1. The results of in vivo biodistribution display rapid and more efficient clearance of 64 Cu-NOTA-rhodamine from the blood and most of the organs and tissues than 64 Cu-NOTAM-rhodamine. A high accumulation by the kidneys (0.85 ± 0.41% ID/g) and a low uptake in the liver (0.35 ± 0.06% ID/g) for 64 Cu-NOTA-rhodamine were observed demonstrating that the main route of Page 7 of 15 AlHokbany et al. EJNMMI Radiopharmacy and Chemistry (2022) 7:19 elimination was the urinary system. Whereas the moderate uptake of 64 Cu-NOTAMrhodamine showed by the liver, spleen, and kidneys (1.19 ± 0.10, 1.19 ± 0.21, and 0.55 ± 0.08% ID/g, respectively) indicates that the route of excretion was predominantly the hepatobiliary and partly urinary systems. Additionally, the main target organ heart displayed the highest uptake of 9.50 ± 0.99% ID/g of the 64 Cu-NOTAM-rhodamine, which is higher than the uptake of the 64 Cu-NOTA-rhodamine conjugate (5.60 ± 1.02% ID/g). Very good heart-to-blood ratios (32.76) and (13.33) were obtained for 64 Cu-NOTAM-and 64 Cu-NOTA-rhodamine conjugates, respectively. Initial Nano-PET imaging studies have clearly delineated the heart uptake of 64 Cu-NOTA-64 Cu-NOTAM-rhodamine conjugates with high contrast relative to the background (Fig. 3). These images are concurrent with findings obtained in quantitative biodistribution data reported above.
These results demonstrate that the 64 Cu-NOTAM-rhodamine conjugate has better heart uptake than the 64 Cu-NOTA-rhodamine conjugate. However, the latter may pose suitable pharmacokinetic properties over the former and deserve more evaluation. Furthermore, the radioactivity excreted into the urine at the time of sacrifice (60 min p.i.) was collected and checked by radio-HPLC to investigate the in vivo stability of 64 Cu-NOTA-and 64 Cu-NOTAM-rhodamine compounds. Radio-HPLC chromatograms of the urine samples displayed that a good amount of radioactivity (> 95%) was still attached to the radiolabeled bioconjugates. These findings show that these radiolabeled bioconjugates are not inclined to fast in vivo degradation and are associated well with the high metabolic stability obtained in human plasma in vitro.

Discussion
In an attempt to develop novel PET rhodamine tracers for MPI studies with longer halflife and better pharmacokinetics, we have developed 64 Cu-NOTA-and 64 Cu-NOTAMrhodamine for myocardial PET imaging. The Cu II ion is a 3d 9 with coordination numbers ranging from 4 through 6 it has borderline hardness with high affinity to O and N donor atoms. Therefore, derivatives of TACN with two (NOTA, NOTAM) carboxymethyl pendant arms both complex Cu(II) with good affinity. The former has an N 3 O 3 donor set that forms a distorted trigonal prismatic geometry. [Cu II NOTA] complex is hexa-coordinated and shows high stability with Log K 21.6. In NOTAM, the presence of more basic donor atoms in the pendant arms has led to an increase in the Log K value of Cu II complexes (Log K 22.4) (Baranyai et al. 2020;Clarke et al. 1990;Clarke and Martell 1991a, b;Wadas et al. 2010;Tolmachev et al. 2017). Therefore, to develop new MPI agents, with suitable characteristics for the PET investigation of myocardial perfusion, we have prepared NOTA-and NOTAM-rhodamine compounds. 64 Cu-Labeled molecules are promising imaging agents for PET due to the favorable nuclear characteristics of the isotope (t 1/2 = 12.7 h, β + 17.4%, E max = 0.656 MeV, β − 39%, E max = 0.573 MeV) and its availability as no-carrier-added Cu-64. The longer physical half-life of 64 Cu compared to other PET isotopes enables imaging at delayed time points, which allows sufficient time for clearance from background tissues, resulting in increased image contrast (Banerjee et al. 2014). The synthetic procedure for the preparation of 64 Cu-NOTA-and 64 Cu-NOTAM-rhodamine provided a facile and simple onestep reaction, with high radiochemical yield and purity.
The results of in vivo biodistribution display rapid and more efficient clearance of 64 Cu-NOTA-rhodamine from the blood and most of the organs and tissues than 64 Cu-NOTAM-rhodamine. A high accumulation by the kidneys and a low uptake in the liver for 64 Cu-NOTA-rhodamine were observed indicating that the urinary system is the main excretion pathway. While 64 Cu-NOTAM-rhodamine showed the moderate uptake by the liver, spleen, and kidneys suggesting that the route of excretion was predominantly the hepatobiliary and partly urinary systems. This behavior may be due to the nature of 64 Cu-NOTA-and 64 Cu-NOTAM as chelating agents and the overall net charge on 64 Cu-NOTA-and 64 Cu-NOTAM-rhodamine molecules (Miao et al. 2006). Additionally, the 64 Cu-NOTAM-rhodamine displayed the highest uptake in the main target organ heart which is higher than the uptake of the 64 Cu-NOTArhodamine conjugate. These values are similar to heart uptake values obtained from other radiofluorinated rhodamine conjugates reported previously ; Bartholoma et al. 2012Bartholoma et al. , 2013Breeman et al. 2005Breeman et al. , 2011 and at least two times better than values obtained using 68 Ga-NOTA-and 68 Ga-NODAGA-rhodamine conjugates. Additionally, good heart-to-blood ratios of 32.76 for 64 .33 for 64 Cu-NOTA-rhodamine conjugates were obtained. The heart-to-blood ratios of the 64 Cu-NOTAM were found to be superior to the values obtained for other rhodamine conjugates, for example, 18 F-FDG-rhodamine (28.10) and 68 Ga-NOTArhodamine (4.56) (Aljammaz et al. 2015a(Aljammaz et al. , b, 2019, suggesting the usefulness of the 64 Cu-NOTAM. Moreover, initial Nano-PET imaging studies have clearly delineated the heart uptake of 64 Cu-NOTA-64 Cu-NOTAM-rhodamine conjugates with high contrast relative to the background (Fig. 3). These images are concurrent with finding obtained in quantitative biodistribution data reported above. The data suggest that the 64 Cu-NOTAM-rhodamine conjugate has better heart uptake properties than the 64 Cu-NOTA-rhodamine conjugate. But, the latter may pose favorable biokinetics over the former and deserve further investigation. Radio-HPLC chromatograms of the urine samples displayed that a good amount of radioactivity was still attached and these radiolabeled bioconjugates are not inclined to fast in vivo degradation. It is worth mentioning here that when these radiolabeled conjugates were investigated in normal Balb/c mice, nearly no accumulation of 64 Cu-NOTA-and 64 Cu-NOTAM-rhodamine in the mice hearts were found, probably due to the in vivo enzymatic breakdown of these radiotracers in mouse serum (data not shown) as observed previously for other rhodamine compounds in mice Aljammaz et al. 2015a, b). These results indicate that mice may not be suitable animal models for the preclinical evaluation of rhodamine conjugates.

Methods
All chemicals and reagents used in this work were all highest purity grade obtained from commercial sources and were used without further purification unless stated. Acetonitrile (ACN) and dimethylformamide (DMF) were kept over molecular sieves. Sep-Pak cartridges were purchased from Waters-Millipore. Thin-layer chromatography-SG sheets were purchased from Grace Discovery Inc. High-performance liquid chromatography (HPLC) analysis was carried out on Luna; Phenomenex C-18 reversed-phase column (analytical, 250 mm × 4.6 mm). The solvent system used was isocratic (eluant: ACN/H2O, 95/5 with 0.1% TFA at a flow rate of 1.0 mL/min). A Jasco chromatographic system equipped with a variable wavelength ultraviolet monitor and in tandem with a Canberra flow through radioactivity detector was used. Ultraviolet absorption was monitored at 254 nm. Chromatograms were acquired and analyzed using BORWIN software. Elemental analyses were performed on a Perkin Elmer CHN 2400 analyzer. The melting points were measured using a Thomas-Hoover Unimelt capillary melting point apparatus. Mass spectroscopy was performed on Quattra electrospray mass spectrometer.

1,4,7-Triazacyclononane-N,N′-diethylamine (NOTAM, 3)
NOTAM was synthesized by dissolving hydrochloric acid salt of 1,4,7-triazacyclononane (TACN. HCl, 500 mg, 2.1 mmol) in ACN (10 mL) followed by the addition of triethylamine (TEA, 1.16 mL, 8.4 mmol) (Scheme 2). To the stirred mixture, bromoethylamine protected with BOC group (430 mg, 2.1 mmol) in ACN (4 mL) was added and the reaction mixture was then stirred and refluxed for 2 h. The solution was concentrated by rotary evaporation to leave a light yellowish solid product which was washed with ACN (10 mL) to leave a yellowish precipitate. This was then filtered, washed with ACN (10 mL), and dissolved in CH 2 Cl 2/ TFA (1:1, 10 mL) before stirring at room temperature for 30 min for deprotection. The mixture was then dried in vacuo to yield 258 mg (57.1%) of NOTAM as a yellow oily material.C 10 H 25 N 5 = MW 215.3.

In vivo biodistribution
Animal studies were conducted strictly according to the international regulations and guidelines governing the safe and proper use of laboratory animals. The biodistribution was carried out in normal Fischer male rats (body weight 50-70 g) to determine the in vivo distribution behavior of the 64 Cu-NOTA-and 64 Cu-NOTAM-rhodamine conjugates. 100 µL of the radiotracers formulated in saline were injected via the lateral tail vein of rats. Each injected dose contained ~ 2.59 MBq of radioactivity. Rats were sacrificed after 1 h post-injection (p.i.) and tissues/organs of interest were dissected, weighed, and counted for radioactivity. The percent of the injected dose per gram (% ID/g) was measured by counting all tissues in a γ-counter.

In vivo nano PET/CT imaging
The PET/CT scans were performed using a preclinical NanoPET/CT scanner (Mediso, Hungary) on normal Fischer male rats (body mass 50-75 g). 64 Cu-NOTAand 64 Cu-NOTAM-rhodamine conjugates (100 μL, 7.4 MBq) were injected into each rat through the tail vein and placed in the NanoPET/CT scanner with continuous O 2 and 2% isoflurane supply. 20 min post tail vein injection of the radiotracers, the rats were imaged for 20 min PET/CT acquisition time. A static scan was acquired at 60 min p.i. The CT scan was performed using the following parameters: X-ray voltage = 50 kVp, Exposure time = 300 ms. A total projection of 288 projects over 360° of rotation was acquired and reconstructed using a cosine filter. This was followed by a PET data acquisition with the following parameters: 5-ns coincidence window and 400-600 keV energy window in 1-5 coincidence mode. Crystal efficiency correction was also applied, with a ring difference of 8, and the images were reconstructed by a three-dimensional ordered-subsets; exception maximum algorithm (subsets, 4; iterations, 6). The pixel size was 0.3 mm. The acquired data in these studies were analyzed by InterVeiw FUSION software developed by Mediso.

Statistical analysis
Data are expressed as mean ± S.D. where appropriate. For data comparisons, a Student's t test was performed on the mean values using Graph-Pad Software (Graph-Pad Software Inc., San Diego, CA, USA). A probability value of P < 0.05 was considered statistically significant.

Conclusion
In a suitable radiosynthesis approach, 64 Cu-NOTA-and 64 Cu-NOTAM-rhodamine compounds were prepared in high radiochemical yields and purities in about 25 min. Preliminary biodistribution in normal Fischer rats at 60 min p.i, exhibited a higher myocardial uptake of 64 Cu-NOTAM-rhodamine conjugate over the 64 Cu-NOTA-rhodamine. The data suggest that these radioconjugates may be suitable for MPI studies using PET. However, further evaluation is needed.