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[68Ga]Ga-HBED-CC-DiAsp: A new renal function imaging agent
Nuclear Medicine and Biology 82–83 (2020) 17–24
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[ 68Ga]Ga-HBED-CC-DiAsp: A new renal function imaging agent Shengyu Shi a, Lifang Zhang a, Zehui Wu b,c, Aili Zhang a, Haiyan Hong a, Seok Rye Choi c, Lin Zhu a,b,⁎, Hank F. Kung b,c,⁎⁎ a b c
Key Laboratory of Radiopharmaceuticals, College of Chemistry, Beijing Normal University, Ministry of Education, Beijing 100875, China Beijing Institute for Brain Disorders, Capital Medical University, Beijing 100069, China Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104, USA
a r t i c l e
i n f o
Article history: Received 30 August 2019 Received in revised form 5 December 2019 Accepted 15 December 2019 Available online xxxx
a b s t r a c t Introduction: [68Ga]Ga-EDTA ([68Ga]Ga-ethylenediaminetetraacetic acid) was previously reported as a renal imaging agent for measuring GFR (glomerular filtration rate). In an effort to provide new agents with better in vivo characteristics for renal imaging, [68Ga]Ga-HBED-CC-DiAsp (Di-Aspartic acid derivative of N,N′-bis [2-hydroxy-5(carboxyethyl)benzyl]-ethylenediamine-N,N′-diacetic acid) was prepared and tested. Method: HBED-CC-DiAsp was synthesized and labeled with [68Ga]GaCl4− at room temperature. Plasma protein and red blood cells (RBC) binding were also evaluated. Biodistribution and dynamic PET imaging studies were performed in mice and rats, respectively. Results: [68Ga]Ga-HBED-CC-DiAsp was radiolabeled at room temperature by a one-step kit formulation in high purity without any purification (radiochemical purity N98%). Previous reports suggested that Ga-HBED-CC exhibited a higher stability constant and rapid chelating formation rate than that of Ga-EDTA (logKGaL = 38.5 vs 22.1, respectively). In vitro stability studies indicated that it was stable up to 120 min. The log DOW value, partition coefficient between n-octanol and water, was found to be −2.52 ± 0.08. Plasma protein and RBC binding was similar to that observed for [68Ga]Ga-EDTA. Biodistribution and dynamic PET/CT imaging studies in rats revealed a rapid clearance primarily through the renal–urinary pathway. The PET-derived [68Ga]Ga-HBED-CC-DiAsp renograms in rats showed an average time-to-peak of 3.6 ± 0.7 min which was similar to that observed for [68Ga]GaEDTA (3.1 ± 0.5 min). The time-to-half-maximal activity was also comparable to that of [68Ga]Ga-EDTA (8.8 vs 8.2 min, respectively). Pretreatment of probenecid, a renal tubular excretion inhibitor, showed no significant effect on renal excretion. Conclusions: [68Ga]Ga-HBED-CC-DiAsp could be prepared quickly at room temperature in high yield and purity. Results of in vitro studies and in vivo biodistribution in mice and rats suggested that [68Ga]Ga-HBED-CC-DiAsp might be useful as a PET imaging agent for measurement of GFR. © 2019 Published by Elsevier Inc.
1. Introduction
Abbreviations: DCM, dichloromethane; DTPA, diethylenetriaminepentaacetic acid; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; EDTA, ethylenediaminetetraacetic acid; FC, flash column chromatography; GFR, glomerulus filtration rate; HBED-CC, N, N′-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N′-diacetic acid; HOBt, 1Hydroxybenzotriazoe; HRMS, High resolution mass spectrometry; ITLC-SG, instant thinlayer chromatography-silica gel; i.v., intravenous; log DOW, n-octanol/water distribution coefficient; NMR, nuclear magnetic resonance; NOTA, 1,4,7-triazacyclononane-triacetic acid; PBS, phosphate buffer saline; PET, positron emission tomography; RBC, red blood cell; RCP, radiochemical purity; ROI, region of interest; RP-HPLC, reversed-phase high performance liquid chromatography; SD rats, Sprague-Dawley rats; SPECT/CT, single-photon emission computed tomography/computed tomography; TFA, trifluoroacetic acid; 2D, 2dimensional; 3D, 3-dimensional. ⁎ Correspondence to: L. Zhu, Key Laboratory of Radiopharmaceuticals, Beijing Normal University, Ministry of Education, No. 19, XinJieKouWai Street, Haidian District, Beijing 100875, China. ⁎⁎ Correspondence to: H. F. Kung, Department of Radiology, University of Pennsylvania, 3700 Market Street, Room 305, Philadelphia, PA 19104, USA. E-mail addresses: [email protected] (L. Zhu), [email protected] (H.F. Kung). https://doi.org/10.1016/j.nucmedbio.2019.12.005 0969-8051/© 2019 Published by Elsevier Inc.
Kidneys are part of major organ systems responsible for various physiologically important functions including waste excretion, fluid regulation, acid-base homeostasis and hormone secretion. One of the standard measurements of renal function used by the urologist and nephrologist is glomerulus filtration rate. Measurement of the renal clearance of inulin employing the constant intravenous infusion technique and bladder catheterization is considered the standard method for the assessment of GFR. However in clinical practice, its use is limited by the high price and time-consuming. Currently, this measurement is performed by plasma sampling and serum creatinine estimation. The method is convenient but it is not very accurate and does not provide any information about individual renal function [1–3]. As an alternative, [ 99mTc]Tc-diethylenetriaminepentaacetic acid 99m ([ Tc]Tc-DTPA) imaging has been applied to determine split GFR estimation by plotting dynamic renograms - time-activity curves
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from planar images from gamma camera. The first path extraction for [ 99mTc]Tc-DTPA was relatively low (20%) compared to previously used glomerular filtration radiotracers: [ 51Cr]Cr-EDTA and [ 125 I] iothalamate, both of which GFR values were measured via plasma sampling without imaging [4,5]. However, in the past few decades, [ 99mTc]Tc-DTPA GFR measurement has been accepted as a method of choice due to its ready availability [4–7]. Precise regional assessment of renal function by [ 99mTc]Tc-DTPA is less accurate due to its quantification is limited by scattering and tissue attenuation [1,4,7]. There are many other 99mTc renal functional imaging agents, which have been used clinically for measuring different renal functions [4,7]. [ 99mTc]Tc-MAG3 (Tubular Secretion) is highly protein-bound and predominantly trapped by proximal renal tubules useful in imaging kidney parenchymal in patients with obstruction and/or impaired renal function. [99mTc]Tc-L,L- and Tc-D,D-ethylenedicysteine (EC) and [99mTc]Tctricarbonylnitriloacetic acid are similarly cleared through tubular secretion for imaging renal function, [ 99mTc]Tc-dimercaptosuccinic Acid (DMSA) is mainly used for imaging cortical retention in the kidney. [ 99mTc]Tc-glucoheptonate (GH) may be useful for measuring cortical retention and GFR imaging. Previously, it was reported that [ 99mTc]Tc-GH is cleared primarily by glomerular filtration, but approximately 10–15% of the injected dose is retained in the renal tubules, allowing delayed, high-resolution static images. These methods have not gained a widespread acceptance for GFR imaging likely due to the lack of specificity in measuring GFR. Recently, increasing availability of PET (positron emission tomography) with superior spatial and temporal resolutions for quantitative tomographic imaging is now being actively employed for measuring GFR [4,7–9]. For this reason, several 18F labeled PET imaging agents specifically have been developed for GFR measurements: including [ 18F] fluorodeoxysorbitol [10–12], and Re(CO)3([ 18F]FEDA) [13]. None of
these 18F GFR imaging agents has been approved by the FDA; currently, they are only being tested in clinical research projects. 68 Ga (T1/2 = 68 min) is attractive as a possible alternative for 18F, because 68Ga is a generator-produced isotope without the need for a nearby cyclotron. Due to the long half-life (T1/2 = 271 days) of parent isotope, 68Ge, the 68Ge/ 68Ga generator would be useful for one year allowing the production of 68Ga PET imaging agents. Another advantage of 68Ga labeled imaging agents using the strong metal chelators, which may be amenable for simple kit formulation. In addition, the flexibility of making 68Ga doses on-site provides unique opportunity for standalone diagnostic imaging clinics. Several commonly used chelating agents, EDTA, DTPA, DOTA, NOTA and HBED-CC, formed stable M(III) complexes. [14–20] (see Fig. 1). They showed very high stability constants for forming Ga 3+ complex (logKGaEDTA = 22.1; logKGaDTPA = 24.3; logKGaDOTA = 21.3; logKGaNOTA = 31.0 and logKGaHBED = 38.5) and relatively high in vivo stability [19–21]. Among them, [ 68Ga]Ga-HBED-CC appeared to be optimal for formation of Ga complexes with the highest stability constant. It may serve as the core structure for developing 68Ga PET imaging agents. Various 68Ga tracers have been reported, EDTA, DTPA, DOTA, and NOTA were labeled with 68Ga [8,22,23]. They all showed favorable renal excretion reflecting GFR. Among them, [ 68Ga]Ga-EDTA has been used in clinical research [23]. Data from preliminary clinical trial indicated that assessment of GFR with [ 68Ga]Ga-EDTA was feasible, with good agreement between plasma sampling and conventional [ 51Cr]CrEDTA. Additional clinical studies suggested [68Ga]Ga-EDTA is a suitable tracer for GFR calculation from PET imaging in humans [9,23,24]. Similarly, [ 68Ga]Ga-NOTA showed high labeling efficiency and low RBC and plasma protein binding in mice. In comparison the GFR values obtained by [ 68Ga]Ga-NOTA and [51Cr]Cr-EDTA were comparable and consistent with those obtained by animal PET imaging studies in mice [22].
Fig. 1. Chemical structures of ligands for complexing Ga3+: DTPA, EDTA, DOTA, NOTA and HBED-CC (logKGaEDTA = 22.1; logKGaDTPA = 24.3; logKGaDOTA = 21.3; logKGaNOTA = 31.0 and logKGaHBED = 38.5) [19–21]. [68Ga]Ga-PSMA 11, containing a [68Ga]Ga-HBED-CC and Lys-Urea-Glu, a PSMA targeting moiety, is the most commonly used 68Ga imaging agent for the diagnosis of prostate cancer tissue over expressing prostate-specific membrane antigen (PSMA) binding sites.
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Previous report suggested that [ 67Ga]Ga-HBED (without the extra carboxylic acids) was substantially excreted into the gastrointestinal tract (67.4%) but there was also appreciable urinary excretion (18.9%) [25]. Later, [ 68Ga]Ga-HBED-CC (with two extra carboxylic acids, see Fig. 1) was used for labeling antibodies [26]. It was found that heterobifunctional agent, HBED-CC-tetrafluorophenol (TFP) ester, was suitable as a rapid conjugating ligand because this agent was forming complexes with [ 68Ga]Ga 3+ much faster than DOTA at ambient temperatures, which would not degrade proteins or peptides [26]. The utility of [ 68Ga]Ga-HBED-CC complex took a very dramatic turn in 2012, when the [ 68Ga]Ga-PSMA 11, containing a [ 68Ga]Ga-HBED-CC complex and a prostate specific membrane antigen (PSMA) targeting Lys-Urea-Glu moiety (Fig. 1), was reported as a suitable imaging for prostate cancer tissues over expressing PSMA binding sites [27–29]. Currently, [ 68Ga] Ga-PSMA 11 is not yet approved by the FDA, but the PSMA11/PET
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imaging has been used in thousands of patients worldwide. It is the most widely used 68Ga imaging agents for diagnosis and monitor prostate cancer [29]. [ 68Ga]Ga-HBED-CC core is highly valuable because it could be prepared quickly at room temperature in high yield with excellent chemical purity. One other highly desirable property of [ 68Ga]Ga-HBED-CC core is that it shows very good in vivo stability, therefore, images obtained usually displayed very low [68Ga]Ga 3+ background activity. It is an excellent starting platform for making new radiopharmaceuticals. Particularly, the [ 68Ga]Ga-HBED-CC labeling could be accomplished using ligand at micrograms quantity without heating. In contrast, milligrams quantity of EDTA and heating was required for [ 68Ga]Ga-EDTA labeling. Taking advantage of these excellent properties of [ 68Ga]GaHBED-CC to further modulate the in vivo kinetic of this complex, two extra aspartic acids (Di-Asp) were introduced to increase hydrophilicity
Scheme 1. Synthesis of HBED-CC-DiAsp (4) and [68Ga]/[natGa]Ga-HBED-CC-DiAsp (5)
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Fig. 2. (A) The radio-HPLC chromatogram of [68Ga]Ga-HBED-CC-DiAsp (B) UV-HPLC chromatogram of natGa-HBED-CC-DiAsp.
leading to rapid renal excretion for GFR measurements. [ 68Ga]GaHBED-CC-DiAsp was prepared and biological evaluations were performed, and results were compared with those of [ 68Ga]Ga-EDTA.
2. Materials and methods 2.1. General Chemicals were purchased from commercial sources and used without further purification. Analytical grade solvents were used without further purification, unless otherwise specified. 68Ge/ 68Ga generator was obtained from ITG (Germany, 20 mCi). HPLC analysis was performed on a Shimadzu LC-20AT 230 V absorbance dual λ detector (Milford, MA, USA) with a reversed-phase column Phenomenex luna C18 150 × 4.6 mm. The analytical conditions were shown in experimental part. HRMS was performed on AB Sciex 5600 Plus. 1H NMR spectra was recorded on a Bruker Advance spectrometer at 400 MHz. A Cobra II autogamma counter (Perkin-Elmer) measured 68Ga radioactivity.
2.2. Chemistry 2.2.1. Synthesis of HBED-CC-DiAsp (4) and Ga-HBED-CC-DiAsp (5) As shown in Scheme 1, a mixture of 1 (322 mg, 0.5 mmol) in 15 mL DCM was added triethylamine (5 mL), N-(3-dimethylaminopropyl)-Nethylcarbodiimide hydrochloride (EDCI, 381 mg, 2 mmol) and 1hydroxybenzotriazole hydrate (HOBt, 270 mg, 2 mmol) at 0 °C. The mixture was stirred at rt. for 1 h. Then L-aspartic acid di-tert-butyl ester hydrochloride (2) (592 mg, 2 mmol) was added into the mixture. The reaction mixture was stirred at rt. for overnight. The solution was washed by H2O, dried over Na2SO4 and filtered. The filtrate was concentrated, and the residue was purified by FC (flash column chromatography, DCM/MeOH/Et3N = 40/1/0.5) to give 327 mg colorless oil 3 (yield: 59.6%). Then trifluoroacetic acid (TFA, 4 mL) was added to above product for 4 h at rt. The solvent was removed under vacuum and the colorless oil 4 was afforded (189 mg, 87.5%). 1H NMR (400 MHz, D6-DMSO) δ: 8.24 (s, 4H), 8.17 (d, 2H, J = 8 Hz), 7.03 (s, 2H), 7.01–6.99 (m, 2H), 6.75 (d, 2H, J = 4 Hz), 4.51–4.47 (m, 4H), 4.13 (d, 2H, J = 4 Hz), 3.97 (s, 4H), 3.60 (s, 4H), 2.79 (d, 2H, J = 4 Hz), 2.53 (m, 2H), 2.46 (m, 4H), 2.34 (t, 4H, J = 6 Hz). HRMS calcd. for C34H42N4O16 [M + H]+ 763.2667; found, 763.2674. 100 mg of 4 (0.13 mmol) was dissolved in 2 mL of DMSO followed by 0.3 mL of GaCl3 (1 mg/mL) in hydrochloric acid (0.05 N) solution, the pH value of the solution was 4–5. The reaction was stirred at rt. for 24 h. The product was extracted with water and ethyl acetate (v/v = 1/1). The aqueous layer was loaded onto an Oasis HLB SPE cartridge (6 cc, 150 mg, conditioned with 10 mL of ethanol followed by 10 mL of H2O), which was then washed with 20 mL H2O. The product was eluted with 2 mL methanol, then the solvent was evaporated to dryness and a white powder 5 was obtained. (yield: 15.9%). 1H NMR (400 MHz, D6DMSO) δ: 12.42 (s, 4H), 8.13 (m, 2H), 7.12 (s, 1H), 6.79 (d, 1H, J = 4 Hz), 6.75 (d, 1H, J = 4 Hz), 6.69 (s, 1H), 6.47 (s, 1H), 6.45 (d, 1H, J = 4 Hz), 4.50 (t, 3H, J = 4 Hz), 4.10–4.04 (m, 4H), 3.77 (m, 2H), 3.57 (m, 2H), 2.85 (m, 2H), 2.69–2.58 (m, 10H), 2.30 (t, 3H, J = 4 Hz). HRMS calcd. for GaC34H40N4O16 [M] + 829.1689; found, 829.1689. 2.3. Radiosynthesis of [ 68Ga]Ga-HBED-CC-DiAsp and [68Ga]Ga-EDTA
Fig. 3. Radiochemical Purity of [68Ga]Ga-HBED-CC-DiAsp from 30 min to 120 min in PBS and plasma. The radiochemical purity of the radiotracer is maintained above 95% up to 120 min both in PBS and in plasma (n = 2).
A 4 mL eluent in 0.05 M HCl of 68Ge/68Ga generator (ITG, Germany) was added to a lyophilized kit which containing 10 μg of HBED-CCDiAsp (0.014 μmol) and 68 mg of NaOAc·3H2O, and incubated at room temperature (25 °C) for 10 min without further purification and the final pH was 4.3. Radiochemical purity (RCP) and radolabeling efficiency were determined using radio-HPLC and radio-TLC. Analytical reversed-phase high performance liquid chromatography (RP-HPLC)
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Fig. 4. Binding of [68Ga]Ga-HBED-CC-DiAsp and [68Ga]Ga-EDTA, respectively, to A erythrocytes (RBCs) or B plasma proteins after 10 or 30 min incubation. n = 3; Binding to rat RBCs of [68Ga]Ga-HBED-CC-DiAsp is 1.3 ± 0.31% at 10 min, 3.5 ± 0.24% at 30 min (p N .05). Binding to rat RBCs of [68Ga]Ga-EDTA is 1.6 ± 0.42% at 10 min, 3.1 ± 0.40% at 30 min. Binding to plasma proteins of [68Ga]Ga-HBED-CC-DiAsp is 3.74 ± 0.45% at 10 min, 4.28 ± 0.56% at 30 min. Binding to plasma proteins of [68Ga]Ga-EDTA is 3.19 ± 0.27% at 10 min, 3.34 ± 0.31% at 30 min (p N .05).
was performed on a Phenomenex luna C18 (5 μm, 150 × 4.6 mm) column using an Shimadzu gradient HPLC System. The [ 68Ga]Ga-HBEDCC-DiAsp was eluted applying different gradients of 0.1% (v/v) TFA in H2O and acetonitrile at a constant flow of 1 mL/min (0–10 min, from 95% H2O with 0.1% TFA to 100% acetonitrile and then back to 95% H2O with 0.1% TFA 15–20 min). ITLC-SG (instant thin-layer chromatography-silica gel, Agilent) developed with 0.2 M NaOAc buffer (pH = 4.5). For the radiosynthesis of [68Ga]Ga-EDTA, 1 mL of the above [ 68Ga]GaCl4− (about 7.4 × 10 3 kBq) eluent was directly added into a 0.5 mL NaOAc buffered solution containing EDTA (10 mg, 34.2 μmol). The solution was heated to 70 °C for 10 min. Quality control was carried out by ITLC-SG which was as same as [ 68Ga]Ga-HBED-CC-DiAsp. After neutralization and sterile filtration, the radiopharmaceuticals can be directly administered to animals. 2.4. In vitro stability in PBS and plasma [ 68Ga]Ga-HBED-CC-DiAsp (740 kBq, 20 μCi) was added to 1 mL phosphate buffered saline (PBS, pH = 7.4, 0.1 M) or human plasma. The solutions were incubated at rt. or 37 °C for 30, 60, 90 and 120 min. Samples were removed and radio-HPLC analysis were performed to measure the stability in PBS and plasma. 2.5. 1-Octanol/water distribution coefficient (log DOW) [ 68Ga]Ga-HBED-CC-DiAsp (100 μL, 3.7 MBq) was added to a mixture of PBS (pH = 7.4): n-octanol (1:1 v/v, 1.9 mL) and the mixture were
shaken vigorously for 5 min at rt. The two layers were separated and the radioactivity associated with 100 μL aliquots of each layer was counted on a Cobra II autogamma counter (Perkin Elmer). The experiment was performed using three separate samples.
2.6. Red blood cell and plasma protein binding [ 68Ga]Ga-HBED-CC-DiAsp and [ 68Ga]Ga-EDTA (740 kBq in 20 μL) were mixed with 100 μL of heparinized blood obtained from rats, respectively and incubated in a shaking incubator at 37 °C. Samples were collected at 10 or 30 min and plasma and blood cell layers were separated by centrifugation at 2000 rpm for 5 min. Blood cells were washed twice with 500 μL of normal saline solution by centrifugation at 2000 rpm for 5 min. The radioactivity of each plasma and blood cell fraction was measured using a gamma counter. The RBCbound fraction of each labeled compound was calculated by obtaining the percentage of RBC radioactivity in the combined fractions. The plasma protein binding measurement was done as follow: 1 mL heparinized plasma samples obtained from rats or an equal volume of PBS were incubated with the radiotracer (370 kBq in 10 μL) for 10 or 30 min. Afterwards, the samples were filtered through a 30-kDa Millipore filter at 1500 ×g for 20 min. The protein bound activity was calculated from the measured activity of the filtrate and the filter membrane taking into account the unspecific binding to the membrane as determined from the PBS samples (≈5% of the total activity).
Table 1 Biodistribution of [68Ga]Ga-EDTA and [68Ga]Ga-HBED-CC-DiAsp at 10 and 30 min after injection in mouse, respectively. Values are expressed as means ± SD of the injected dose (740 kBq) normalized per gram tissue (n = 5). Organ
[68Ga]Ga-HBED-CC-DiAsp
[68Ga]Ga-EDTA
10 min (probenecid)
10 min
30 min
Heart Lung Liver Spleen Kidney Stomach Intestine Muscle Blood
1.85 ± 0.45 3.54 ± 0.87 1.45 ± 0.52⁎ 1.35 ± 0.71 10.77 ± 2.55 0.77 ± 0.24 1.03 ± 0.25 1.61 ± 0.62 5.70 ± 1.10
1.68 ± 0.31 3.63 ± 0.63 2.54 ± 0.27† 1.72 ± 0.56 12.51 ± 3.18 0.64 ± 0.14† 1.33 ± 0.20 1.82 ± 0.26 5.69 ± 1.53
0.80 2.08 2.09 2.41 5.51 0.82 0.63 0.85 2.46
⁎ p b .05 (comparing the %ID/g between 10 min (probenecid) and 10 min for [68Ga]Ga-HBED-CC-DiAsp). † p b .05 (comparing the %ID/g between [68Ga]Ga-HBED-CC-DiAsp and [68Ga]Ga-EDTA at 10 min). †† p b .05 (comparing the %ID/g between [68Ga]Ga-HBED-CC-DiAsp and [68Ga]Ga-EDTA at 30 min).
± ± ± ± ± ± ± ± ±
0.13 0.43 0.30†† 0.60†† 1.60 0.39 0.09 0.10†† 0.49
10 min
30 min
1.54 ± 0.14 3.74 ± 0.49 1.20 ± 0.18 1.14 ± 0.11 11.27 ± 2.91 1.51 ± 0.35 1.17 ± 0.04 2.05 ± 0.57 4.78 ± 0.70
0.56 1.44 0.59 0.62 5.44 0.77 0.73 0.52 1.67
± ± ± ± ± ± ± ± ±
0.13 0.27 0.13 0.07 0.43 0.19 0.39 0.05 0.36
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Fig. 5. Results of in vivo [68Ga]Ga-HBED-CC-DiAsp PET imaging in a rat. A: renal dynamic PET images of [68Ga]Ga-HBED-CC-DiAsp from 0 min to 50 min (SUV-bw, SUV-body weight). B: Time-activity curves of the kidneys and bladder measured by dynamic PET imaging. A separate experiment was performed with a rat pretreated with probenecid. C: 3D PET-CT fusion images of [68Ga]Ga-HBED-CC-DiAsp at 3 min and 50 min.
2.7. Biodistribution and intake mechanism study Male Kunming mice (18–22 g) were injected with radiotracer (740 kBq, 0.1 mL) through a tail vein under anesthesia with 2% isoflurane. The injected mice were killed at 10 and 30 min postinjection. Blood, liver, muscle, kidney, lung, heart, intestine, stomach and spleen were then excised, blotted, and weighed, and then 68Ga radioactivity of each organ was counted by a gamma scintillation counter. Results were expressed as the percentage of injected dose per gram of tissue (%ID/g). The mechanism of excretion of [ 68Ga]Ga-HBED-CCDiAsp in kidney was investigated by injecting probenecid (renal tubular excretion inhibitor) prior to the injection of [ 68Ga]Ga-HBED-CCDiAsp. 100 mg of probenecid was dissolved in 5 mL of water. The pH was adjusted to 8.0 with 0.1 M NaOH. Each mouse was injected with 0.1 mL probenecid solution 10 min before the injection of radiotracer. The injected mice were killed at 10 min post-injection, and same operation was performed as above.
5–10 min 30 s one frame (10 frames × 30 s), 10–50 min 2 min one frame (20 frames × 2 min), 50–60 min 5 min one frame (2 frames × 5 min) (n = 3) Regions of interest (ROIs) were drawn over kidneys guided by CT images using Amira 3.1 image visualization and analysis software. Radioactivity within the ROIs of each frame was calculated. The radioactivity in the kidney ROI for each frame was decaycorrected. The time-activity curve of both kidneys (renogram) was plotted from the kidney ROI activities in each frame. Renogram analysis was performed for each rat. Time-to-peak activity and time to half-maximal activity values were averaged for left and right kidneys for each rat. 2.9. Statistical analysis The significance of the differences among values was estimated by SPSS software (Version 20.0), p b .05 was considered statistically significant. All of the values were expressed as the mean ± the standard deviation (SD).
2.8. Small animal PET/CT imaging 3. Results Dynamic PET/CT imaging studies were conducted in healthy male Sprague Dawley rats (n = 3, 190–210 g) on Siemens Inveon. Rats were anesthetized using 2% isoflurane in oxygen at 2 L/min, in an induction chamber. When fully anesthetized, the animals were placed on the scanner bed, with a nose cone used to maintain anesthesia at 1% isoflurane in oxygen at 2 L/min. Body temperature was maintained at 37 °C by using a water-circulated pad under the animals. Immediately after the start of the scan, a dose of radiotracer (~500 μCi, 18.5 MBq) was injected via the tail vein. To investigate the effect of probenecid on renal excretion of two labeled compounds in rats, 20 mg of probenecid was injected via the tail vein 10 min before the PET scan. Dynamic PET imaging data in list-mode were acquired over a period of 60 min after injection followed by acquiring CT imaging data over a period of 5–10 min. The PET data were reconstructed using a 2D filtered back projection (FBP) algorithm. 0–5 min 10 s one frame (30 frames × 10 s),
3.1. Chemistry In order to obtain a renal imaging agent with simple labeling conditions and high renal uptake, we combined the chelator HBCD-CC with two aspartic acid groups. The target compound is expected to rapidly and efficiently label 68Ga 3+ at room temperature and displaying high renal uptake and excretion. We obtained HBED-CC derivative, 1, by a reported synthesis method [27]. The compound 1 and the L-aspartic acid di-tert-butyl ester are subjected to a condensation reaction to obtain intermediate compound 3. After treatment of 3 with TFA overnight, the tbutyl ester protecting groups were removed to give the desired precursor 4. GaCl3 and precursor, 4, were stirred at room temperature to form the “cold” Ga complex 5. The chemical structures of compound 4 and 5 were confirmed by NMR and HRMS-ESI. The HRMS-ESI, showed the
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expected molecular ion mass, while the peak patterns matched the theoretical isotope distribution. 3.2. Radiosynthesis After mixing [ 68Ga]GaCl4− with EDTA and HBED-CC-DiAsp the labeling efficiency of both 68Ga conjugates were N95%, as confirmed by radioTLC. The preparation of [ 68Ga]Ga-HBED-CC-DiAsp was carried out at rt. and 10 μg of precursor was enough. Radiolabeling experiments for [ 68Ga]Ga-HBED-CC-DiAsp were performed 27 times at room temperature with an average radiolabeling efficiency of 97.2 ± 1.7%. However, heating was needed and 10 mg of EDTA was required to ensure the radiolabeling efficiency of [ 68Ga]Ga-EDTA reaching N95% labeling yields. Radiolabeling experiments for [ 68Ga]Ga-EDTA were carried out 16 times and gave an average of radiolabeling yield of 95.6 ± 2.1%. Radio-TLC with NaOAc buffer showed Rf = 0 for 68Ga colloid and free of [ 68Ga]GaCl4− and Rf = 0.9–1.0 for radiotracers. The radio-HPLC chromatogram of [68Ga]Ga-HBED-CC-DiAsp was shown in Fig. 2A, the retention time is 7.07 min, RCP is N98%. When [68Ga]Ga-HBED-CC-DiAsp and nat Ga-HBED-CC-DiAsp were co-injected in HPLC, the radioactivity (7.07 min) and UV peak (6.84 min) (Fig. 2A and B) showed similar peak shape and retention time, which provided strong evidence for confirmation. A small delay in retention time on HPLC between UV and radioactivity signal was due to a sequential connection of the detectors. The RCP of [ 68Ga]Ga-EDTA was measured in the same way as [ 68Ga] Ga-HBED-CC-DiAsp, which was also N98%. 3.3. In vitro stability in PBS and plasma The RCP was measured and analyzed by the radio-HPLC to determine the stability of radiotracer as a function of time, which was shown in Fig. 3. Results suggested that [68Ga]Ga-HBED-CC-DiAsp maintained high stability in both PBS and plasma up to 120 min. 3.4. n-Octanol/water distribution coefficient (log DOW) The log DOW value determined at pH = 7.4 of [ 68Ga]Ga-HBED-CCDiAsp was −2.52 ± 0.08. This showed that the radiotracer has strong water solubility. 3.5. RBC and plasma protein binding As shown in Fig. 4, [ 68Ga]Ga-HBED-CC-DiAsp demonstrated similar binding (1.3 ± 0.3%, at 10 min) to rat RBCs as [ 68Ga]Ga-EDTA (1.6 ± 0.4%, at 10 min). These data indicated that [ 68Ga]Ga-HBED-CC-DiAsp showed sufficiently low binding to RBCs, and thus might be used as glomerular function imaging agent. [ 68Ga]Ga-HBED-CC-DiAsp showed a slightly more binding to plasma proteins (3.74 ± 0.45%, at 10 min) than [ 68Ga]Ga-EDTA (3.19 ± 0.27%, at 10 min). 3.6. Biodistribution and intake mechanism Biodistribution studies in male Kunming mice after tail vein injection using [ 68Ga]Ga-EDTA and [ 68Ga]Ga-HBED-CC-DiAsp was performed and the results were shown in Table 1. Two radiotracers showed very similar biodistribution patterns. High kidney uptakes were found at 10 min and decreased rapidly at 30 min. For [ 68Ga]GaHBED-CC-DiAsp the uptake of kidney was 12.51 ± 3.18%ID/g at 10 min and it was reduced rapidly to 5.51 ± 1.60%ID/g at 30 min. For [ 68Ga]Ga-EDTA, it was reduced from 11.27 ± 2.91%ID/g to 5.44 ± 0.43%ID/g. The second highest radioactivity was found in the blood. For [ 68Ga]Ga-HBED-CC-DiAsp, it was 5.69 ± 1.53%ID/g and decreased rapidly to 2.46 ± 0.49%ID/g at 30 min. Both agents showed low liver and stomach uptake. For [68Ga]Ga-HBED-CC-DiAsp, the injection of probenecid showed no significant effect on the renal excretion at 10 min.
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3.7. Small animal PET/CT imaging Dynamic animal-PET/CT imaging of a rat during 60 min after tail vein injection of [ 68Ga]Ga-EDTA and [ 68Ga]Ga-HBED-CC-DiAsp revealed a rapid clearance primarily through the renal–urinary pathway, which was shown in Fig. 5. The whole body's 3D PET-CT fusion images showed that the uptake of this tracer in other organs, such as heart, liver, and intestine, were very low compared with kidney and bladder. Total activity counts for left and right kidneys were obtained from animal-PET images for quantitative analysis. The PET-derived [68Ga]Ga-HBED-CC-DiAsp renograms revealed an average time-to-peak of 3.6 ± 0.7 min which was similar to that of [68Ga]Ga-EDTA (3.1 ± 0.5 min), and the average timeto-half-maximal activity (8.8 ± 1.0 min) was also found to be similar to that of [ 68Ga]Ga-EDTA (8.2 ± 1.1 min). Renograms showed that pretreatment of probenecid in a rat had no significant effect on renal excretion (Fig. 5B).
4. Discussion A multitude of radiology imaging methods including traditional radio-graphic techniques, scintigraphy with nuclear medicine agents, ultrasonography, computed tomography (CT), magnetic resonance imaging (MRI), single photon emission computer tomography (SPECT) and positron emission tomography (PET) are available [30]. In additional to using [ 99mTc]Tc-DTPA for dynamic renal scintigraphy measuring GFR, other radionuclide renal imaging agents are available. The most commonly used agents include [ 99m Tc]Tcdimercaptosuccinic acid ([ 99mTc]Tc-DMSA) for imaging renal tubular secretion, and similarly, [ 99m Tc]Tc-mercaptoacetyltriglycine ([ 99mTc]Tc-MAG3) was often used for mapping renal parenchymal imaging [4,5,7,30]. In this paper it was demonstrated that [ 68Ga]Ga-HBED-CC-DiAsp could be prepared by a simple and rapid radiosynthesis method with high radiochemical purity (RCP N 95%) in room temperature using a kit formulation. It was found that a kit containing merely 10 micrograms of precursor (HBED-CC-DiAsp, 3.3 nm/mL) was sufficient for complex [ 68Ga]Ga 3+ without heating, which was conveniently performed as compared to that reported for radiolabeling [ 68 Ga]GaEDTA or [ 68Ga]Ga-NOTA (200 nm/mL of ligand was needed for the radiolabeling of these two radiotracers at room temperature [22]). [ 68Ga]Ga-HBED-CC-DiAsp was stable over 120 min in either PBS or human plasma at 37 °C. Low logDOW value of [ 68Ga]Ga-HBED-CCDiAsp showed that it was highly water soluble and suitable as a kidney imaging agent. Low red blood cell or plasma protein binding also suggested that it might be a good renal imaging agent, because the low binding would likely increase the filtration rate through the glomeruli into urine. [ 68 Ga]Ga-HBED-CC-DiAsp showed comparable RBC or plasma protein binding with that of [ 68Ga]Ga-EDTA suggesting that it would be a potential PET imaging agent for GFR. Biodistribution studies show that, both radiotracers have high renal uptake at 10 min, and it was reduced N50% until 30 min. In addition, the second highest radioactivity was found in the blood and it decreased rapidly at 30 min equally. Both agents showed measurable uptakes in the lungs, which might actually correspond to residual blood radioactivity in the lung. Probenecid would competitively inhibit the excretion of weak organic acids in the renal tubules. Prior injection of probenecid showed no significant effect on the renal excretion at 10 min suggesting a minimum tubular excretion, which indicated that the glomerular filtration and excretion was the predominant mechanism for [ 68Ga]Ga-HBED-CC-DiAsp. Results described above suggested that [ 68Ga]Ga-HBED-CC-DiAsp displayed similar rapid glomerular filtration comparable to that of [ 68Ga]GaEDTA. The kit formulation for preparing [ 68Ga]Ga-HBED-CC-DiAsp could provide a GFR imaging readily available for routine clinical PET imaging without the need of a nearby cyclotron.
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5. Conclusion [ 68Ga]Ga-HBED-CC-DiAsp could be prepared at room temperature in high yield and excellent radiochemical purity via a simple kit formulation. In vivo and in vitro evaluations and dynamic PET/CT imaging studies suggested that [ 68Ga]Ga-HBED-CC-DiAsp may be potentially useful as a PET imaging agent for measurement of GFR. Acknowledgements This work was supported in part by grants from the National Key Research and Development Program of China (2016YFC1306300), Beijing Science and Technology Planning Project (Z151100003915116) and Key Field Research and Development Program of Guangdong Province (2018B030337001). References [1] Blaufox MD. PET measurement of renal glomerular filtration rate: is there a role in nuclear medicine? J Nucl Med 2016;57:1495–6. [2] Inker LA, Schmid CH, Tighiouart H, Eckfeldt JH, Feldman HI, Greene T, et al. Estimating glomerular filtration rate from serum creatinine and cystatin C. N Engl J Med 2012;367:20–9. [3] Blaufox MD, De Palma D, Taylor A, Szabo Z, Prigent A, Samal M, et al. The SNMMI and EANM practice guideline for renal scintigraphy in adults. Eur J Nucl Med Mol Imaging 2018;45:2218–28. [4] Taylor AT. Radionuclides in nephrourology, part 2: pitfalls and diagnostic applications. J Nucl Med 2014;55:786–98. [5] Taylor AT. Radionuclides in nephrourology, part 1: radiopharmaceuticals, quality control, and quantitative indices. J Nucl Med 2014;55:608–15. [6] Blaufox MD. Renal background correction and measurement of split renal function: the challenge: editorial comment: EJNM-D-15-00322, M Donald Blaufox, MD, PhD. Eur J Nucl Med Mol Imaging 2016;43:548–9. [7] Taylor AT, Garcia EV. Computer-assisted diagnosis in renal nuclear medicine: rationale, methodology, and interpretative criteria for diuretic renography. Semin Nucl Med 2014;44:146–58. [8] Werner RA, Chen X, Lapa C, Koshino K, Rowe SP, Pomper MG, et al. The next era of renal radionuclide imaging: novel PET radiotracers. Eur J Nucl Med Mol Imaging 2019;46:1773–86. [9] Hofman M, Binns D, Johnston V, Siva S, Thompson M, Eu P, et al. 68Ga-EDTA PET/CT imaging and plasma clearance for glomerular filtration rate quantification: comparison to conventional 51Cr-EDTA. J Nucl Med 2015;56:405–9. [10] Werner RA, Wakabayashi H, Chen X, Hirano M, Shinaji T, Lapa C, et al. Functional renal imaging with 2-deoxy-2-18F-fluorosorbitol PET in rat models of renal disorders. J Nucl Med 2018;59:828–32. [11] Wakabayashi H, Werner RA, Hayakawa N, Javadi MS, Xinyu C, Herrmann K, et al. Initial preclinical evaluation of 18F-fluorodeoxysorbitol PET as a novel functional renal imaging agent. J Nucl Med 2016;57:1625–8.
[12] Werner RA, Ordonez AA, Sanchez-Bautista J, Marcus C, Lapa C, Rowe SP, et al. Novel functional renal PET imaging with 18F-FDS in human subjects. Clin Nucl Med 2019; 44:410–1. [13] Lipowska M, Jarkas N, Voll RJ, Nye JA, Klenc J, Goodman MM, et al. Re(CO)3([18F] FEDA), a novel 18F PET renal tracer: radiosynthesis and preclinical evaluation. Nucl Med Biol 2018;58:42–50. [14] Motekaitis RJ, Martell AE, Welch MJ. Stability of trivalent metal complexes of phenolic acids related to N,N’-bis(2-hydroxylbenzyl)-N,N’-diacetic acid (HBED). Inorg Chem 1990;29:1463–7. [15] Motekaitis R, Sun Y, Martell A. New synthetic, selective, high-affinity ligands for effective trivalent metal-ion binding and transport. Inorg Chim Acta 1992;198–200: 421–8. [16] Viola NA, Rarig Jr RS, Ouellette W, Doyle RP. Synthesis, structure and thermal analysis of the gallium complex of 1,4,7,10-tetraazacyclo-dodecane-N,N′,N″,N‴tetraacetic acid (DOTA). Polyhedron 2006;25:3457–62. [17] Burke BP, Archibald SJ. Macrocyclic coordination chemistry. Annu Rep Prog Chem, Sect A: Inorg Chem 2013;109:232–53. [18] Burke BP, Clemente GS, Archibald SJ. Recent advances in chelator design and labelling methodology for 68Ga radiopharmaceuticals. J Labelled Comp Radiopharm 2014;57:239–43. [19] Price EW, Orvig C. Matching chelators to radiometals for radiopharmaceuticals. Chem Soc Rev 2014;43:260–90. [20] Price TW, Greenman J, Stasiuk GJ. Current advances in ligand design for inorganic positron emission tomography tracers 68Ga, 64Cu, 89Zr and 44Sc. Dalton Trans 2016;45:15702–24. [21] Wadas TJ, Wong EH, Weisman GR, Anderson CJ. Coordinating radiometals of copper, gallium, indium, yttrium, and zirconium for PET and SPECT imaging of disease. Chem Rev 2010;110:2858–902. [22] Lee JY, Jeong JM, Kim YJ, Jeong HJ, Lee YS, Lee DS, et al. Preparation of Ga-68-NOTA as a renal PET agent and feasibility tests in mice. Nucl Med Biol 2014;41:210–5. [23] Gundel D, Pohle U, Prell E, Odparlik A, Thews O. Assessing glomerular filtration in small animals using [68Ga]DTPA and [68Ga]EDTA with PET imaging. Mol Imaging Biol 2018;20:457–64. [24] Hofman MS, Hicks RJ. Gallium-68 EDTA PET/CT for renal imaging. Semin Nucl Med 2016;46:448–61. [25] Hunt FC. Potential radiopharmaceuticals for the diagnosis of biliary atresia and neonatal hepatitis: EHPG and HBED chelates of 67Ga and 111In. Int J Rad Appl Instrum B 1988;15:659–64. [26] Eder M, Wangler B, Knackmuss S, LeGall F, Little M, Haberkorn U, et al. Tetrafluorophenolate of HBED-CC: a versatile conjugation agent for 68Ga-labeled small recombinant antibodies. Eur J Nucl Med Mol Imaging 2008;35:1878–86. [27] Eder M, Schafer M, Bauder-Wust U, Hull WE, Wangler C, Mier W, et al. 68Ga-complex lipophilicity and the targeting property of a urea-based PSMA inhibitor for PET imaging. Bioconjug Chem 2012;23:688–97. [28] Hofman MS, Hicks RJ, Maurer T, Eiber M. Prostate-specific membrane antigen PET: clinical utility in prostate cancer, normal patterns, pearls, and pitfalls. Radiographics 2018;38:200–17. [29] Rowe SP, Gorin MA, Pomper MG. Imaging of prostate-specific membrane antigen with small-molecule PET radiotracers: from the bench to advanced clinical applications. Annu Rev Med 2019;70:461–77. [30] Fried JG, Morgan MA. Renal imaging: core curriculum 2019. Am J Kidney Dis 2019; 73:552–65.
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[ 68Ga]Ga-HBED-CC-DiAsp: A new renal function imaging agent Shengyu Shi a, Lifang Zhang a, Zehui Wu b,c, Aili Zhang a, Haiyan Hong a, Seok Rye Choi c, Lin Zhu a,b,⁎, Hank F. Kung b,c,⁎⁎ a b c
Key Laboratory of Radiopharmaceuticals, College of Chemistry, Beijing Normal University, Ministry of Education, Beijing 100875, China Beijing Institute for Brain Disorders, Capital Medical University, Beijing 100069, China Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104, USA
a r t i c l e
i n f o
Article history: Received 30 August 2019 Received in revised form 5 December 2019 Accepted 15 December 2019 Available online xxxx
a b s t r a c t Introduction: [68Ga]Ga-EDTA ([68Ga]Ga-ethylenediaminetetraacetic acid) was previously reported as a renal imaging agent for measuring GFR (glomerular filtration rate). In an effort to provide new agents with better in vivo characteristics for renal imaging, [68Ga]Ga-HBED-CC-DiAsp (Di-Aspartic acid derivative of N,N′-bis [2-hydroxy-5(carboxyethyl)benzyl]-ethylenediamine-N,N′-diacetic acid) was prepared and tested. Method: HBED-CC-DiAsp was synthesized and labeled with [68Ga]GaCl4− at room temperature. Plasma protein and red blood cells (RBC) binding were also evaluated. Biodistribution and dynamic PET imaging studies were performed in mice and rats, respectively. Results: [68Ga]Ga-HBED-CC-DiAsp was radiolabeled at room temperature by a one-step kit formulation in high purity without any purification (radiochemical purity N98%). Previous reports suggested that Ga-HBED-CC exhibited a higher stability constant and rapid chelating formation rate than that of Ga-EDTA (logKGaL = 38.5 vs 22.1, respectively). In vitro stability studies indicated that it was stable up to 120 min. The log DOW value, partition coefficient between n-octanol and water, was found to be −2.52 ± 0.08. Plasma protein and RBC binding was similar to that observed for [68Ga]Ga-EDTA. Biodistribution and dynamic PET/CT imaging studies in rats revealed a rapid clearance primarily through the renal–urinary pathway. The PET-derived [68Ga]Ga-HBED-CC-DiAsp renograms in rats showed an average time-to-peak of 3.6 ± 0.7 min which was similar to that observed for [68Ga]GaEDTA (3.1 ± 0.5 min). The time-to-half-maximal activity was also comparable to that of [68Ga]Ga-EDTA (8.8 vs 8.2 min, respectively). Pretreatment of probenecid, a renal tubular excretion inhibitor, showed no significant effect on renal excretion. Conclusions: [68Ga]Ga-HBED-CC-DiAsp could be prepared quickly at room temperature in high yield and purity. Results of in vitro studies and in vivo biodistribution in mice and rats suggested that [68Ga]Ga-HBED-CC-DiAsp might be useful as a PET imaging agent for measurement of GFR. © 2019 Published by Elsevier Inc.
1. Introduction
Abbreviations: DCM, dichloromethane; DTPA, diethylenetriaminepentaacetic acid; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; EDTA, ethylenediaminetetraacetic acid; FC, flash column chromatography; GFR, glomerulus filtration rate; HBED-CC, N, N′-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N′-diacetic acid; HOBt, 1Hydroxybenzotriazoe; HRMS, High resolution mass spectrometry; ITLC-SG, instant thinlayer chromatography-silica gel; i.v., intravenous; log DOW, n-octanol/water distribution coefficient; NMR, nuclear magnetic resonance; NOTA, 1,4,7-triazacyclononane-triacetic acid; PBS, phosphate buffer saline; PET, positron emission tomography; RBC, red blood cell; RCP, radiochemical purity; ROI, region of interest; RP-HPLC, reversed-phase high performance liquid chromatography; SD rats, Sprague-Dawley rats; SPECT/CT, single-photon emission computed tomography/computed tomography; TFA, trifluoroacetic acid; 2D, 2dimensional; 3D, 3-dimensional. ⁎ Correspondence to: L. Zhu, Key Laboratory of Radiopharmaceuticals, Beijing Normal University, Ministry of Education, No. 19, XinJieKouWai Street, Haidian District, Beijing 100875, China. ⁎⁎ Correspondence to: H. F. Kung, Department of Radiology, University of Pennsylvania, 3700 Market Street, Room 305, Philadelphia, PA 19104, USA. E-mail addresses: [email protected] (L. Zhu), [email protected] (H.F. Kung). https://doi.org/10.1016/j.nucmedbio.2019.12.005 0969-8051/© 2019 Published by Elsevier Inc.
Kidneys are part of major organ systems responsible for various physiologically important functions including waste excretion, fluid regulation, acid-base homeostasis and hormone secretion. One of the standard measurements of renal function used by the urologist and nephrologist is glomerulus filtration rate. Measurement of the renal clearance of inulin employing the constant intravenous infusion technique and bladder catheterization is considered the standard method for the assessment of GFR. However in clinical practice, its use is limited by the high price and time-consuming. Currently, this measurement is performed by plasma sampling and serum creatinine estimation. The method is convenient but it is not very accurate and does not provide any information about individual renal function [1–3]. As an alternative, [ 99mTc]Tc-diethylenetriaminepentaacetic acid 99m ([ Tc]Tc-DTPA) imaging has been applied to determine split GFR estimation by plotting dynamic renograms - time-activity curves
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from planar images from gamma camera. The first path extraction for [ 99mTc]Tc-DTPA was relatively low (20%) compared to previously used glomerular filtration radiotracers: [ 51Cr]Cr-EDTA and [ 125 I] iothalamate, both of which GFR values were measured via plasma sampling without imaging [4,5]. However, in the past few decades, [ 99mTc]Tc-DTPA GFR measurement has been accepted as a method of choice due to its ready availability [4–7]. Precise regional assessment of renal function by [ 99mTc]Tc-DTPA is less accurate due to its quantification is limited by scattering and tissue attenuation [1,4,7]. There are many other 99mTc renal functional imaging agents, which have been used clinically for measuring different renal functions [4,7]. [ 99mTc]Tc-MAG3 (Tubular Secretion) is highly protein-bound and predominantly trapped by proximal renal tubules useful in imaging kidney parenchymal in patients with obstruction and/or impaired renal function. [99mTc]Tc-L,L- and Tc-D,D-ethylenedicysteine (EC) and [99mTc]Tctricarbonylnitriloacetic acid are similarly cleared through tubular secretion for imaging renal function, [ 99mTc]Tc-dimercaptosuccinic Acid (DMSA) is mainly used for imaging cortical retention in the kidney. [ 99mTc]Tc-glucoheptonate (GH) may be useful for measuring cortical retention and GFR imaging. Previously, it was reported that [ 99mTc]Tc-GH is cleared primarily by glomerular filtration, but approximately 10–15% of the injected dose is retained in the renal tubules, allowing delayed, high-resolution static images. These methods have not gained a widespread acceptance for GFR imaging likely due to the lack of specificity in measuring GFR. Recently, increasing availability of PET (positron emission tomography) with superior spatial and temporal resolutions for quantitative tomographic imaging is now being actively employed for measuring GFR [4,7–9]. For this reason, several 18F labeled PET imaging agents specifically have been developed for GFR measurements: including [ 18F] fluorodeoxysorbitol [10–12], and Re(CO)3([ 18F]FEDA) [13]. None of
these 18F GFR imaging agents has been approved by the FDA; currently, they are only being tested in clinical research projects. 68 Ga (T1/2 = 68 min) is attractive as a possible alternative for 18F, because 68Ga is a generator-produced isotope without the need for a nearby cyclotron. Due to the long half-life (T1/2 = 271 days) of parent isotope, 68Ge, the 68Ge/ 68Ga generator would be useful for one year allowing the production of 68Ga PET imaging agents. Another advantage of 68Ga labeled imaging agents using the strong metal chelators, which may be amenable for simple kit formulation. In addition, the flexibility of making 68Ga doses on-site provides unique opportunity for standalone diagnostic imaging clinics. Several commonly used chelating agents, EDTA, DTPA, DOTA, NOTA and HBED-CC, formed stable M(III) complexes. [14–20] (see Fig. 1). They showed very high stability constants for forming Ga 3+ complex (logKGaEDTA = 22.1; logKGaDTPA = 24.3; logKGaDOTA = 21.3; logKGaNOTA = 31.0 and logKGaHBED = 38.5) and relatively high in vivo stability [19–21]. Among them, [ 68Ga]Ga-HBED-CC appeared to be optimal for formation of Ga complexes with the highest stability constant. It may serve as the core structure for developing 68Ga PET imaging agents. Various 68Ga tracers have been reported, EDTA, DTPA, DOTA, and NOTA were labeled with 68Ga [8,22,23]. They all showed favorable renal excretion reflecting GFR. Among them, [ 68Ga]Ga-EDTA has been used in clinical research [23]. Data from preliminary clinical trial indicated that assessment of GFR with [ 68Ga]Ga-EDTA was feasible, with good agreement between plasma sampling and conventional [ 51Cr]CrEDTA. Additional clinical studies suggested [68Ga]Ga-EDTA is a suitable tracer for GFR calculation from PET imaging in humans [9,23,24]. Similarly, [ 68Ga]Ga-NOTA showed high labeling efficiency and low RBC and plasma protein binding in mice. In comparison the GFR values obtained by [ 68Ga]Ga-NOTA and [51Cr]Cr-EDTA were comparable and consistent with those obtained by animal PET imaging studies in mice [22].
Fig. 1. Chemical structures of ligands for complexing Ga3+: DTPA, EDTA, DOTA, NOTA and HBED-CC (logKGaEDTA = 22.1; logKGaDTPA = 24.3; logKGaDOTA = 21.3; logKGaNOTA = 31.0 and logKGaHBED = 38.5) [19–21]. [68Ga]Ga-PSMA 11, containing a [68Ga]Ga-HBED-CC and Lys-Urea-Glu, a PSMA targeting moiety, is the most commonly used 68Ga imaging agent for the diagnosis of prostate cancer tissue over expressing prostate-specific membrane antigen (PSMA) binding sites.
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Previous report suggested that [ 67Ga]Ga-HBED (without the extra carboxylic acids) was substantially excreted into the gastrointestinal tract (67.4%) but there was also appreciable urinary excretion (18.9%) [25]. Later, [ 68Ga]Ga-HBED-CC (with two extra carboxylic acids, see Fig. 1) was used for labeling antibodies [26]. It was found that heterobifunctional agent, HBED-CC-tetrafluorophenol (TFP) ester, was suitable as a rapid conjugating ligand because this agent was forming complexes with [ 68Ga]Ga 3+ much faster than DOTA at ambient temperatures, which would not degrade proteins or peptides [26]. The utility of [ 68Ga]Ga-HBED-CC complex took a very dramatic turn in 2012, when the [ 68Ga]Ga-PSMA 11, containing a [ 68Ga]Ga-HBED-CC complex and a prostate specific membrane antigen (PSMA) targeting Lys-Urea-Glu moiety (Fig. 1), was reported as a suitable imaging for prostate cancer tissues over expressing PSMA binding sites [27–29]. Currently, [ 68Ga] Ga-PSMA 11 is not yet approved by the FDA, but the PSMA11/PET
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imaging has been used in thousands of patients worldwide. It is the most widely used 68Ga imaging agents for diagnosis and monitor prostate cancer [29]. [ 68Ga]Ga-HBED-CC core is highly valuable because it could be prepared quickly at room temperature in high yield with excellent chemical purity. One other highly desirable property of [ 68Ga]Ga-HBED-CC core is that it shows very good in vivo stability, therefore, images obtained usually displayed very low [68Ga]Ga 3+ background activity. It is an excellent starting platform for making new radiopharmaceuticals. Particularly, the [ 68Ga]Ga-HBED-CC labeling could be accomplished using ligand at micrograms quantity without heating. In contrast, milligrams quantity of EDTA and heating was required for [ 68Ga]Ga-EDTA labeling. Taking advantage of these excellent properties of [ 68Ga]GaHBED-CC to further modulate the in vivo kinetic of this complex, two extra aspartic acids (Di-Asp) were introduced to increase hydrophilicity
Scheme 1. Synthesis of HBED-CC-DiAsp (4) and [68Ga]/[natGa]Ga-HBED-CC-DiAsp (5)
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Fig. 2. (A) The radio-HPLC chromatogram of [68Ga]Ga-HBED-CC-DiAsp (B) UV-HPLC chromatogram of natGa-HBED-CC-DiAsp.
leading to rapid renal excretion for GFR measurements. [ 68Ga]GaHBED-CC-DiAsp was prepared and biological evaluations were performed, and results were compared with those of [ 68Ga]Ga-EDTA.
2. Materials and methods 2.1. General Chemicals were purchased from commercial sources and used without further purification. Analytical grade solvents were used without further purification, unless otherwise specified. 68Ge/ 68Ga generator was obtained from ITG (Germany, 20 mCi). HPLC analysis was performed on a Shimadzu LC-20AT 230 V absorbance dual λ detector (Milford, MA, USA) with a reversed-phase column Phenomenex luna C18 150 × 4.6 mm. The analytical conditions were shown in experimental part. HRMS was performed on AB Sciex 5600 Plus. 1H NMR spectra was recorded on a Bruker Advance spectrometer at 400 MHz. A Cobra II autogamma counter (Perkin-Elmer) measured 68Ga radioactivity.
2.2. Chemistry 2.2.1. Synthesis of HBED-CC-DiAsp (4) and Ga-HBED-CC-DiAsp (5) As shown in Scheme 1, a mixture of 1 (322 mg, 0.5 mmol) in 15 mL DCM was added triethylamine (5 mL), N-(3-dimethylaminopropyl)-Nethylcarbodiimide hydrochloride (EDCI, 381 mg, 2 mmol) and 1hydroxybenzotriazole hydrate (HOBt, 270 mg, 2 mmol) at 0 °C. The mixture was stirred at rt. for 1 h. Then L-aspartic acid di-tert-butyl ester hydrochloride (2) (592 mg, 2 mmol) was added into the mixture. The reaction mixture was stirred at rt. for overnight. The solution was washed by H2O, dried over Na2SO4 and filtered. The filtrate was concentrated, and the residue was purified by FC (flash column chromatography, DCM/MeOH/Et3N = 40/1/0.5) to give 327 mg colorless oil 3 (yield: 59.6%). Then trifluoroacetic acid (TFA, 4 mL) was added to above product for 4 h at rt. The solvent was removed under vacuum and the colorless oil 4 was afforded (189 mg, 87.5%). 1H NMR (400 MHz, D6-DMSO) δ: 8.24 (s, 4H), 8.17 (d, 2H, J = 8 Hz), 7.03 (s, 2H), 7.01–6.99 (m, 2H), 6.75 (d, 2H, J = 4 Hz), 4.51–4.47 (m, 4H), 4.13 (d, 2H, J = 4 Hz), 3.97 (s, 4H), 3.60 (s, 4H), 2.79 (d, 2H, J = 4 Hz), 2.53 (m, 2H), 2.46 (m, 4H), 2.34 (t, 4H, J = 6 Hz). HRMS calcd. for C34H42N4O16 [M + H]+ 763.2667; found, 763.2674. 100 mg of 4 (0.13 mmol) was dissolved in 2 mL of DMSO followed by 0.3 mL of GaCl3 (1 mg/mL) in hydrochloric acid (0.05 N) solution, the pH value of the solution was 4–5. The reaction was stirred at rt. for 24 h. The product was extracted with water and ethyl acetate (v/v = 1/1). The aqueous layer was loaded onto an Oasis HLB SPE cartridge (6 cc, 150 mg, conditioned with 10 mL of ethanol followed by 10 mL of H2O), which was then washed with 20 mL H2O. The product was eluted with 2 mL methanol, then the solvent was evaporated to dryness and a white powder 5 was obtained. (yield: 15.9%). 1H NMR (400 MHz, D6DMSO) δ: 12.42 (s, 4H), 8.13 (m, 2H), 7.12 (s, 1H), 6.79 (d, 1H, J = 4 Hz), 6.75 (d, 1H, J = 4 Hz), 6.69 (s, 1H), 6.47 (s, 1H), 6.45 (d, 1H, J = 4 Hz), 4.50 (t, 3H, J = 4 Hz), 4.10–4.04 (m, 4H), 3.77 (m, 2H), 3.57 (m, 2H), 2.85 (m, 2H), 2.69–2.58 (m, 10H), 2.30 (t, 3H, J = 4 Hz). HRMS calcd. for GaC34H40N4O16 [M] + 829.1689; found, 829.1689. 2.3. Radiosynthesis of [ 68Ga]Ga-HBED-CC-DiAsp and [68Ga]Ga-EDTA
Fig. 3. Radiochemical Purity of [68Ga]Ga-HBED-CC-DiAsp from 30 min to 120 min in PBS and plasma. The radiochemical purity of the radiotracer is maintained above 95% up to 120 min both in PBS and in plasma (n = 2).
A 4 mL eluent in 0.05 M HCl of 68Ge/68Ga generator (ITG, Germany) was added to a lyophilized kit which containing 10 μg of HBED-CCDiAsp (0.014 μmol) and 68 mg of NaOAc·3H2O, and incubated at room temperature (25 °C) for 10 min without further purification and the final pH was 4.3. Radiochemical purity (RCP) and radolabeling efficiency were determined using radio-HPLC and radio-TLC. Analytical reversed-phase high performance liquid chromatography (RP-HPLC)
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21
Fig. 4. Binding of [68Ga]Ga-HBED-CC-DiAsp and [68Ga]Ga-EDTA, respectively, to A erythrocytes (RBCs) or B plasma proteins after 10 or 30 min incubation. n = 3; Binding to rat RBCs of [68Ga]Ga-HBED-CC-DiAsp is 1.3 ± 0.31% at 10 min, 3.5 ± 0.24% at 30 min (p N .05). Binding to rat RBCs of [68Ga]Ga-EDTA is 1.6 ± 0.42% at 10 min, 3.1 ± 0.40% at 30 min. Binding to plasma proteins of [68Ga]Ga-HBED-CC-DiAsp is 3.74 ± 0.45% at 10 min, 4.28 ± 0.56% at 30 min. Binding to plasma proteins of [68Ga]Ga-EDTA is 3.19 ± 0.27% at 10 min, 3.34 ± 0.31% at 30 min (p N .05).
was performed on a Phenomenex luna C18 (5 μm, 150 × 4.6 mm) column using an Shimadzu gradient HPLC System. The [ 68Ga]Ga-HBEDCC-DiAsp was eluted applying different gradients of 0.1% (v/v) TFA in H2O and acetonitrile at a constant flow of 1 mL/min (0–10 min, from 95% H2O with 0.1% TFA to 100% acetonitrile and then back to 95% H2O with 0.1% TFA 15–20 min). ITLC-SG (instant thin-layer chromatography-silica gel, Agilent) developed with 0.2 M NaOAc buffer (pH = 4.5). For the radiosynthesis of [68Ga]Ga-EDTA, 1 mL of the above [ 68Ga]GaCl4− (about 7.4 × 10 3 kBq) eluent was directly added into a 0.5 mL NaOAc buffered solution containing EDTA (10 mg, 34.2 μmol). The solution was heated to 70 °C for 10 min. Quality control was carried out by ITLC-SG which was as same as [ 68Ga]Ga-HBED-CC-DiAsp. After neutralization and sterile filtration, the radiopharmaceuticals can be directly administered to animals. 2.4. In vitro stability in PBS and plasma [ 68Ga]Ga-HBED-CC-DiAsp (740 kBq, 20 μCi) was added to 1 mL phosphate buffered saline (PBS, pH = 7.4, 0.1 M) or human plasma. The solutions were incubated at rt. or 37 °C for 30, 60, 90 and 120 min. Samples were removed and radio-HPLC analysis were performed to measure the stability in PBS and plasma. 2.5. 1-Octanol/water distribution coefficient (log DOW) [ 68Ga]Ga-HBED-CC-DiAsp (100 μL, 3.7 MBq) was added to a mixture of PBS (pH = 7.4): n-octanol (1:1 v/v, 1.9 mL) and the mixture were
shaken vigorously for 5 min at rt. The two layers were separated and the radioactivity associated with 100 μL aliquots of each layer was counted on a Cobra II autogamma counter (Perkin Elmer). The experiment was performed using three separate samples.
2.6. Red blood cell and plasma protein binding [ 68Ga]Ga-HBED-CC-DiAsp and [ 68Ga]Ga-EDTA (740 kBq in 20 μL) were mixed with 100 μL of heparinized blood obtained from rats, respectively and incubated in a shaking incubator at 37 °C. Samples were collected at 10 or 30 min and plasma and blood cell layers were separated by centrifugation at 2000 rpm for 5 min. Blood cells were washed twice with 500 μL of normal saline solution by centrifugation at 2000 rpm for 5 min. The radioactivity of each plasma and blood cell fraction was measured using a gamma counter. The RBCbound fraction of each labeled compound was calculated by obtaining the percentage of RBC radioactivity in the combined fractions. The plasma protein binding measurement was done as follow: 1 mL heparinized plasma samples obtained from rats or an equal volume of PBS were incubated with the radiotracer (370 kBq in 10 μL) for 10 or 30 min. Afterwards, the samples were filtered through a 30-kDa Millipore filter at 1500 ×g for 20 min. The protein bound activity was calculated from the measured activity of the filtrate and the filter membrane taking into account the unspecific binding to the membrane as determined from the PBS samples (≈5% of the total activity).
Table 1 Biodistribution of [68Ga]Ga-EDTA and [68Ga]Ga-HBED-CC-DiAsp at 10 and 30 min after injection in mouse, respectively. Values are expressed as means ± SD of the injected dose (740 kBq) normalized per gram tissue (n = 5). Organ
[68Ga]Ga-HBED-CC-DiAsp
[68Ga]Ga-EDTA
10 min (probenecid)
10 min
30 min
Heart Lung Liver Spleen Kidney Stomach Intestine Muscle Blood
1.85 ± 0.45 3.54 ± 0.87 1.45 ± 0.52⁎ 1.35 ± 0.71 10.77 ± 2.55 0.77 ± 0.24 1.03 ± 0.25 1.61 ± 0.62 5.70 ± 1.10
1.68 ± 0.31 3.63 ± 0.63 2.54 ± 0.27† 1.72 ± 0.56 12.51 ± 3.18 0.64 ± 0.14† 1.33 ± 0.20 1.82 ± 0.26 5.69 ± 1.53
0.80 2.08 2.09 2.41 5.51 0.82 0.63 0.85 2.46
⁎ p b .05 (comparing the %ID/g between 10 min (probenecid) and 10 min for [68Ga]Ga-HBED-CC-DiAsp). † p b .05 (comparing the %ID/g between [68Ga]Ga-HBED-CC-DiAsp and [68Ga]Ga-EDTA at 10 min). †† p b .05 (comparing the %ID/g between [68Ga]Ga-HBED-CC-DiAsp and [68Ga]Ga-EDTA at 30 min).
± ± ± ± ± ± ± ± ±
0.13 0.43 0.30†† 0.60†† 1.60 0.39 0.09 0.10†† 0.49
10 min
30 min
1.54 ± 0.14 3.74 ± 0.49 1.20 ± 0.18 1.14 ± 0.11 11.27 ± 2.91 1.51 ± 0.35 1.17 ± 0.04 2.05 ± 0.57 4.78 ± 0.70
0.56 1.44 0.59 0.62 5.44 0.77 0.73 0.52 1.67
± ± ± ± ± ± ± ± ±
0.13 0.27 0.13 0.07 0.43 0.19 0.39 0.05 0.36
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Fig. 5. Results of in vivo [68Ga]Ga-HBED-CC-DiAsp PET imaging in a rat. A: renal dynamic PET images of [68Ga]Ga-HBED-CC-DiAsp from 0 min to 50 min (SUV-bw, SUV-body weight). B: Time-activity curves of the kidneys and bladder measured by dynamic PET imaging. A separate experiment was performed with a rat pretreated with probenecid. C: 3D PET-CT fusion images of [68Ga]Ga-HBED-CC-DiAsp at 3 min and 50 min.
2.7. Biodistribution and intake mechanism study Male Kunming mice (18–22 g) were injected with radiotracer (740 kBq, 0.1 mL) through a tail vein under anesthesia with 2% isoflurane. The injected mice were killed at 10 and 30 min postinjection. Blood, liver, muscle, kidney, lung, heart, intestine, stomach and spleen were then excised, blotted, and weighed, and then 68Ga radioactivity of each organ was counted by a gamma scintillation counter. Results were expressed as the percentage of injected dose per gram of tissue (%ID/g). The mechanism of excretion of [ 68Ga]Ga-HBED-CCDiAsp in kidney was investigated by injecting probenecid (renal tubular excretion inhibitor) prior to the injection of [ 68Ga]Ga-HBED-CCDiAsp. 100 mg of probenecid was dissolved in 5 mL of water. The pH was adjusted to 8.0 with 0.1 M NaOH. Each mouse was injected with 0.1 mL probenecid solution 10 min before the injection of radiotracer. The injected mice were killed at 10 min post-injection, and same operation was performed as above.
5–10 min 30 s one frame (10 frames × 30 s), 10–50 min 2 min one frame (20 frames × 2 min), 50–60 min 5 min one frame (2 frames × 5 min) (n = 3) Regions of interest (ROIs) were drawn over kidneys guided by CT images using Amira 3.1 image visualization and analysis software. Radioactivity within the ROIs of each frame was calculated. The radioactivity in the kidney ROI for each frame was decaycorrected. The time-activity curve of both kidneys (renogram) was plotted from the kidney ROI activities in each frame. Renogram analysis was performed for each rat. Time-to-peak activity and time to half-maximal activity values were averaged for left and right kidneys for each rat. 2.9. Statistical analysis The significance of the differences among values was estimated by SPSS software (Version 20.0), p b .05 was considered statistically significant. All of the values were expressed as the mean ± the standard deviation (SD).
2.8. Small animal PET/CT imaging 3. Results Dynamic PET/CT imaging studies were conducted in healthy male Sprague Dawley rats (n = 3, 190–210 g) on Siemens Inveon. Rats were anesthetized using 2% isoflurane in oxygen at 2 L/min, in an induction chamber. When fully anesthetized, the animals were placed on the scanner bed, with a nose cone used to maintain anesthesia at 1% isoflurane in oxygen at 2 L/min. Body temperature was maintained at 37 °C by using a water-circulated pad under the animals. Immediately after the start of the scan, a dose of radiotracer (~500 μCi, 18.5 MBq) was injected via the tail vein. To investigate the effect of probenecid on renal excretion of two labeled compounds in rats, 20 mg of probenecid was injected via the tail vein 10 min before the PET scan. Dynamic PET imaging data in list-mode were acquired over a period of 60 min after injection followed by acquiring CT imaging data over a period of 5–10 min. The PET data were reconstructed using a 2D filtered back projection (FBP) algorithm. 0–5 min 10 s one frame (30 frames × 10 s),
3.1. Chemistry In order to obtain a renal imaging agent with simple labeling conditions and high renal uptake, we combined the chelator HBCD-CC with two aspartic acid groups. The target compound is expected to rapidly and efficiently label 68Ga 3+ at room temperature and displaying high renal uptake and excretion. We obtained HBED-CC derivative, 1, by a reported synthesis method [27]. The compound 1 and the L-aspartic acid di-tert-butyl ester are subjected to a condensation reaction to obtain intermediate compound 3. After treatment of 3 with TFA overnight, the tbutyl ester protecting groups were removed to give the desired precursor 4. GaCl3 and precursor, 4, were stirred at room temperature to form the “cold” Ga complex 5. The chemical structures of compound 4 and 5 were confirmed by NMR and HRMS-ESI. The HRMS-ESI, showed the
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expected molecular ion mass, while the peak patterns matched the theoretical isotope distribution. 3.2. Radiosynthesis After mixing [ 68Ga]GaCl4− with EDTA and HBED-CC-DiAsp the labeling efficiency of both 68Ga conjugates were N95%, as confirmed by radioTLC. The preparation of [ 68Ga]Ga-HBED-CC-DiAsp was carried out at rt. and 10 μg of precursor was enough. Radiolabeling experiments for [ 68Ga]Ga-HBED-CC-DiAsp were performed 27 times at room temperature with an average radiolabeling efficiency of 97.2 ± 1.7%. However, heating was needed and 10 mg of EDTA was required to ensure the radiolabeling efficiency of [ 68Ga]Ga-EDTA reaching N95% labeling yields. Radiolabeling experiments for [ 68Ga]Ga-EDTA were carried out 16 times and gave an average of radiolabeling yield of 95.6 ± 2.1%. Radio-TLC with NaOAc buffer showed Rf = 0 for 68Ga colloid and free of [ 68Ga]GaCl4− and Rf = 0.9–1.0 for radiotracers. The radio-HPLC chromatogram of [68Ga]Ga-HBED-CC-DiAsp was shown in Fig. 2A, the retention time is 7.07 min, RCP is N98%. When [68Ga]Ga-HBED-CC-DiAsp and nat Ga-HBED-CC-DiAsp were co-injected in HPLC, the radioactivity (7.07 min) and UV peak (6.84 min) (Fig. 2A and B) showed similar peak shape and retention time, which provided strong evidence for confirmation. A small delay in retention time on HPLC between UV and radioactivity signal was due to a sequential connection of the detectors. The RCP of [ 68Ga]Ga-EDTA was measured in the same way as [ 68Ga] Ga-HBED-CC-DiAsp, which was also N98%. 3.3. In vitro stability in PBS and plasma The RCP was measured and analyzed by the radio-HPLC to determine the stability of radiotracer as a function of time, which was shown in Fig. 3. Results suggested that [68Ga]Ga-HBED-CC-DiAsp maintained high stability in both PBS and plasma up to 120 min. 3.4. n-Octanol/water distribution coefficient (log DOW) The log DOW value determined at pH = 7.4 of [ 68Ga]Ga-HBED-CCDiAsp was −2.52 ± 0.08. This showed that the radiotracer has strong water solubility. 3.5. RBC and plasma protein binding As shown in Fig. 4, [ 68Ga]Ga-HBED-CC-DiAsp demonstrated similar binding (1.3 ± 0.3%, at 10 min) to rat RBCs as [ 68Ga]Ga-EDTA (1.6 ± 0.4%, at 10 min). These data indicated that [ 68Ga]Ga-HBED-CC-DiAsp showed sufficiently low binding to RBCs, and thus might be used as glomerular function imaging agent. [ 68Ga]Ga-HBED-CC-DiAsp showed a slightly more binding to plasma proteins (3.74 ± 0.45%, at 10 min) than [ 68Ga]Ga-EDTA (3.19 ± 0.27%, at 10 min). 3.6. Biodistribution and intake mechanism Biodistribution studies in male Kunming mice after tail vein injection using [ 68Ga]Ga-EDTA and [ 68Ga]Ga-HBED-CC-DiAsp was performed and the results were shown in Table 1. Two radiotracers showed very similar biodistribution patterns. High kidney uptakes were found at 10 min and decreased rapidly at 30 min. For [ 68Ga]GaHBED-CC-DiAsp the uptake of kidney was 12.51 ± 3.18%ID/g at 10 min and it was reduced rapidly to 5.51 ± 1.60%ID/g at 30 min. For [ 68Ga]Ga-EDTA, it was reduced from 11.27 ± 2.91%ID/g to 5.44 ± 0.43%ID/g. The second highest radioactivity was found in the blood. For [ 68Ga]Ga-HBED-CC-DiAsp, it was 5.69 ± 1.53%ID/g and decreased rapidly to 2.46 ± 0.49%ID/g at 30 min. Both agents showed low liver and stomach uptake. For [68Ga]Ga-HBED-CC-DiAsp, the injection of probenecid showed no significant effect on the renal excretion at 10 min.
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3.7. Small animal PET/CT imaging Dynamic animal-PET/CT imaging of a rat during 60 min after tail vein injection of [ 68Ga]Ga-EDTA and [ 68Ga]Ga-HBED-CC-DiAsp revealed a rapid clearance primarily through the renal–urinary pathway, which was shown in Fig. 5. The whole body's 3D PET-CT fusion images showed that the uptake of this tracer in other organs, such as heart, liver, and intestine, were very low compared with kidney and bladder. Total activity counts for left and right kidneys were obtained from animal-PET images for quantitative analysis. The PET-derived [68Ga]Ga-HBED-CC-DiAsp renograms revealed an average time-to-peak of 3.6 ± 0.7 min which was similar to that of [68Ga]Ga-EDTA (3.1 ± 0.5 min), and the average timeto-half-maximal activity (8.8 ± 1.0 min) was also found to be similar to that of [ 68Ga]Ga-EDTA (8.2 ± 1.1 min). Renograms showed that pretreatment of probenecid in a rat had no significant effect on renal excretion (Fig. 5B).
4. Discussion A multitude of radiology imaging methods including traditional radio-graphic techniques, scintigraphy with nuclear medicine agents, ultrasonography, computed tomography (CT), magnetic resonance imaging (MRI), single photon emission computer tomography (SPECT) and positron emission tomography (PET) are available [30]. In additional to using [ 99mTc]Tc-DTPA for dynamic renal scintigraphy measuring GFR, other radionuclide renal imaging agents are available. The most commonly used agents include [ 99m Tc]Tcdimercaptosuccinic acid ([ 99mTc]Tc-DMSA) for imaging renal tubular secretion, and similarly, [ 99m Tc]Tc-mercaptoacetyltriglycine ([ 99mTc]Tc-MAG3) was often used for mapping renal parenchymal imaging [4,5,7,30]. In this paper it was demonstrated that [ 68Ga]Ga-HBED-CC-DiAsp could be prepared by a simple and rapid radiosynthesis method with high radiochemical purity (RCP N 95%) in room temperature using a kit formulation. It was found that a kit containing merely 10 micrograms of precursor (HBED-CC-DiAsp, 3.3 nm/mL) was sufficient for complex [ 68Ga]Ga 3+ without heating, which was conveniently performed as compared to that reported for radiolabeling [ 68 Ga]GaEDTA or [ 68Ga]Ga-NOTA (200 nm/mL of ligand was needed for the radiolabeling of these two radiotracers at room temperature [22]). [ 68Ga]Ga-HBED-CC-DiAsp was stable over 120 min in either PBS or human plasma at 37 °C. Low logDOW value of [ 68Ga]Ga-HBED-CCDiAsp showed that it was highly water soluble and suitable as a kidney imaging agent. Low red blood cell or plasma protein binding also suggested that it might be a good renal imaging agent, because the low binding would likely increase the filtration rate through the glomeruli into urine. [ 68 Ga]Ga-HBED-CC-DiAsp showed comparable RBC or plasma protein binding with that of [ 68Ga]Ga-EDTA suggesting that it would be a potential PET imaging agent for GFR. Biodistribution studies show that, both radiotracers have high renal uptake at 10 min, and it was reduced N50% until 30 min. In addition, the second highest radioactivity was found in the blood and it decreased rapidly at 30 min equally. Both agents showed measurable uptakes in the lungs, which might actually correspond to residual blood radioactivity in the lung. Probenecid would competitively inhibit the excretion of weak organic acids in the renal tubules. Prior injection of probenecid showed no significant effect on the renal excretion at 10 min suggesting a minimum tubular excretion, which indicated that the glomerular filtration and excretion was the predominant mechanism for [ 68Ga]Ga-HBED-CC-DiAsp. Results described above suggested that [ 68Ga]Ga-HBED-CC-DiAsp displayed similar rapid glomerular filtration comparable to that of [ 68Ga]GaEDTA. The kit formulation for preparing [ 68Ga]Ga-HBED-CC-DiAsp could provide a GFR imaging readily available for routine clinical PET imaging without the need of a nearby cyclotron.
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5. Conclusion [ 68Ga]Ga-HBED-CC-DiAsp could be prepared at room temperature in high yield and excellent radiochemical purity via a simple kit formulation. In vivo and in vitro evaluations and dynamic PET/CT imaging studies suggested that [ 68Ga]Ga-HBED-CC-DiAsp may be potentially useful as a PET imaging agent for measurement of GFR. Acknowledgements This work was supported in part by grants from the National Key Research and Development Program of China (2016YFC1306300), Beijing Science and Technology Planning Project (Z151100003915116) and Key Field Research and Development Program of Guangdong Province (2018B030337001). References [1] Blaufox MD. PET measurement of renal glomerular filtration rate: is there a role in nuclear medicine? J Nucl Med 2016;57:1495–6. [2] Inker LA, Schmid CH, Tighiouart H, Eckfeldt JH, Feldman HI, Greene T, et al. Estimating glomerular filtration rate from serum creatinine and cystatin C. N Engl J Med 2012;367:20–9. [3] Blaufox MD, De Palma D, Taylor A, Szabo Z, Prigent A, Samal M, et al. The SNMMI and EANM practice guideline for renal scintigraphy in adults. Eur J Nucl Med Mol Imaging 2018;45:2218–28. [4] Taylor AT. Radionuclides in nephrourology, part 2: pitfalls and diagnostic applications. J Nucl Med 2014;55:786–98. [5] Taylor AT. Radionuclides in nephrourology, part 1: radiopharmaceuticals, quality control, and quantitative indices. J Nucl Med 2014;55:608–15. [6] Blaufox MD. Renal background correction and measurement of split renal function: the challenge: editorial comment: EJNM-D-15-00322, M Donald Blaufox, MD, PhD. Eur J Nucl Med Mol Imaging 2016;43:548–9. [7] Taylor AT, Garcia EV. Computer-assisted diagnosis in renal nuclear medicine: rationale, methodology, and interpretative criteria for diuretic renography. Semin Nucl Med 2014;44:146–58. [8] Werner RA, Chen X, Lapa C, Koshino K, Rowe SP, Pomper MG, et al. The next era of renal radionuclide imaging: novel PET radiotracers. Eur J Nucl Med Mol Imaging 2019;46:1773–86. [9] Hofman M, Binns D, Johnston V, Siva S, Thompson M, Eu P, et al. 68Ga-EDTA PET/CT imaging and plasma clearance for glomerular filtration rate quantification: comparison to conventional 51Cr-EDTA. J Nucl Med 2015;56:405–9. [10] Werner RA, Wakabayashi H, Chen X, Hirano M, Shinaji T, Lapa C, et al. Functional renal imaging with 2-deoxy-2-18F-fluorosorbitol PET in rat models of renal disorders. J Nucl Med 2018;59:828–32. [11] Wakabayashi H, Werner RA, Hayakawa N, Javadi MS, Xinyu C, Herrmann K, et al. Initial preclinical evaluation of 18F-fluorodeoxysorbitol PET as a novel functional renal imaging agent. J Nucl Med 2016;57:1625–8.
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