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Basic Science Investigation |
1 ICIS–CNR, Corso Stati Uniti, Padova, Italy; 2 Department of Pharmaceutical Sciences, University of Padua, Padova, Italy; 3 Research Center, Nihon Medi-Physics Co., Ltd., Chiba, Japan; and 4 Nuclear Medicine Service, Istituto Oncologico Veneto (IOV), Padova, Italy
Correspondence: For correspondence contact: Cristina Bolzati, ICIS, CNR Corso Stati Uniti, 4, 35127 Padova, Italy. E-mail: bolzati{at}icis.cnr.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Key Words: DBODC • myocardial imaging agent • technetium • metabolism • MDR
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Comprehension of the biologic factors determining the pharmacokinetic behavior of these agents is crucial not only to elucidate their diagnostic importance but also to provide a rational design strategy aimed at improving the biologic properties.
This study was addressed to elucidate the mechanisms of distribution, retention, and excretion of the new cardiac 99mTc(N)-agents. The sex-related in vitro and in vivo stability and subcellular distribution of 99mTc(N)-DBODC(3/5) as representatives of this class of compounds were investigated. Data collected for 99mTc(N)-DBODC(3) were used for comparison and to confirm the metabolic pattern of 99mTc(N)-DBODC(5). The effect of cyclosporin A on the pharmacokinetic properties was also investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analyses were used to evaluate the stability of the compounds as radiochemical purity. TLC analyses were performed on C18 F254S plates (Merck) using a mixture of saline/CH3OH/THF/HAc(g) (2/8/1/1) as mobile phase (99mTc(N)-DBODC(3): Rf = 0.43; 99mTc(N)-DBODC(5): Rf = 0.61) and on SiO2 F254 plates (Merck) using a mixture of EtOH/CHCl3/Tol/NH4Ac (0.5 mol dm–1) (5/3/3/1) (99mTc(N)-DBODC(3): Rf = 0.78; 99mTc(N)-DBODC(5): Rf = 0.80). The activity on the plates was detected using a Cyclone phosphorus imaging instrument (Packard).
HPLC analysis were performed on a System Gold instrument (Beckman) equipped with programmable solvent model 126, sample injection valve 210A, scanning detector module 166, and radioisotope detector model B-FC-3200 (Bioscan). HPLC analysis was performed using a reversed-phase Beckman octadecyl silane precolumn (4.6 x 45 mm, 5 µm), and a reversed-phase Beckman octadecyl silane column (4.6 x 250 mm, 5 µm). Isocratic: solvent A = phosphate buffer (0.02 mol dm–1; pH 7.4) and solvent B = MeOH (20:80); flow rate = 1 mL/min for 30 min; ultraviolet 
= 215 nm; 99mTc(N)-DBODC(3) tr = 13.83 min and 99mTc(N)-DBODC(5) tr = 18.53 min. Serum protein binding affinity was evaluated through chromatographic methods using HPLC or a size-exclusion MicroSpin G50 column (Amersham Biosciences). Reversed-phase HPLC was performed with a Symmetry 300 C4 precolumn (3.9 x 20 mm, 5 µm; Waters) and a Symmetry 300 C4 column (4.6 x 150 mm, 5 µm). Gradient: solvent A = H2O (0.1% trifluoroacetic acid, pH 3) and solvent B = CH3CN (0.1% trifluoroacetic acid) (0–2 min, 15% B; 2–20 min, 65% B; 20–22 min, 15% B; 22–25 min, 15% B); flow rate = 1 mL/min.
99mTc(N)-DBODC(3/5) Radiopharmaceutical Preparation
These agents were prepared in accordance with published procedures (3). Before use, the complexes were purified with a Sep-Pak C18 cartridge (Waters) conditioned with 5 mL of EtOH and 5 mL of deionized water. The reaction solution containing the 99mTc(N)-complex was diluted with 8.0 mL of deionized water and loaded on the cartridge. Approximately 95% of the initial activity was retained on the cartridge. After washing the cartridge with water (20.0 mL) and ethanol, 35% (5.0 mL), the complex was eluted using an 80/20 mixture of ethanol/saline (0.250 mL x 1; 0.750 mL x 1). Ninety percent of the loaded activity was collected in the second fraction. The solvents were evaporated under a dinitrogen stream; the complex was dissolved in water to obtain an isotonic saline solution containing less than 5% (v/v) ethanol and used for in vitro and in vivo studies.
The radiochemical purity of each compound, determined by TLC and HPLC techniques, was more than 98%.
Determination of Log P and Log K0' Values
Log P values were determined by vortex mixing (20 min) 3 mL of n-octanol, 3 mL of phosphate buffer (0.02 mol dm–1, pH 7.4), and 100 µL of the 99mTc(N)-radiolabeled compounds purified by HPLC chromatography and treated with a Sep-Pak C18 cartridge as indicated above. After centrifugation (3,000g for 10 min), aliquots (100 µL; in triplets) of both the organic and the aqueous phases were collected and counted with a 
-counter. The P values were calculated using the following equation: P = (activity concentration in n-octanol)/(activity concentration in aqueous solution).
Log K0' values of the radiolabeled compounds were measured using the previously reported method (3). For comparison, the log P and log K0' values of 99mTc-sestamibi were determined.
In Vitro Studies
Protein Binding.
The affinity of the 99mTc(N)-complexes toward the serum proteins was evaluated by chromatographic methods, using the procedures reported below.
In a propylene test tube, 100 µL of the purified complex (50–100 MBq) were added to 900 µL of human or rat serum; at 2, 10, and 60 min of incubation at 37°C, 150 µL of each sample were withdrawn and diluted in 1.350 mL of phosphate buffer (phosphate-buffered saline, 0.02 mol dm–1, pH 7.4). Aliquots of this solution were treated and analyzed. In method A, 100 µL were analyzed by HPLC, without further purification, using a reversed-phase Symmetry C4 column. In method B, 25 µL were loaded on a prespun (735g for 1 min) size-exclusion MicroSpin G50 column). The column was centrifuged at 735g for 1 min. The collected eluate and the column were counted in a -counter. The protein-bound complex was calculated as the percentage of the total activity. Twenty-five microliters of 99mTc-complex were controlled after incubation in phosphate-buffered saline at 37°C as a blank. As a control, a small fraction of the collected eluate was analyzed by HPLC. The chromatographic profiles revealed the presence of only the protein fraction. All activity was associated with the mini column.
Metabolism.
For incubation in liver homogenates, the organs freshly excised from female and male rats were rapidly rinsed and homogenized in 20 mM HEPES buffer (pH 7.3) with an Ultra-Turrax T25 homogenator (IKA Works, Inc.) for 1 min at 4°C.
The in vitro metabolism of the agents was evaluated by monitoring the radiochemical purity at different time points using the following procedure. In a propylene test tube, 50 µL of the purified 99mTc-complex (50–100 MBq) were added to 950 µL of rat serum, 950 µL of human serum, 950 µL of fresh 30% liver homogenate, and 950 µL of saline and HEPES buffer as a blank. The samples were incubated at 37°C for 24 h. Aliquots (200 µL) of each solution were withdrawn, diluted with 800 µL of phosphate buffer (0.2 mol dm–1, pH 7.4), and treated using an OASIS hydrophilic lipophilic balance (HLB) extraction cartridge (Waters) before HPLC injection. In detail, the sample was loaded on cartridges, conditioned with MeOH (1 mL), equilibrated with water (1 mL), and washed with MeOH 5% (3 mL). Ninety percent of the starting activity was eluted using a 90/10 mixture of EtOH and saline (1 mL). Aliquots of this fraction were analyzed by TLC and HPLC.
Animal Studies
Animal experiments were performed in compliance with the relevant national laws relating to the conduct of animal experimentation. Studies were performed on both female and male Sprague–Dawley rats.
Biodistribution.
The animals were anesthetized with an intraperitoneal injection of a mixture of zolazepam HCl plus tiletamine HCl (Zoletil; Virbac [40 mg kg–1]) and xylazine (2 mg kg–1). A 150-µL dose of the radioactive complex (300–370 kBq) was injected through the jugular vein. At different times after injection, animals (n = 3) were sacrificed. A blood sample and the excised organs rinsed with saline were weighed and counted in a -counter. Results were normalized as percentage injected dose per gram.
In Vivo Stability.
The complexes were extracted from the rat tissues and urine using the following procedure. A 150-µL dose of radioactive compound (370–500 MBq) was injected through the jugular vein in anesthetized rats. At 30 and 120 min after injection, animals (n = 3) were sacrificed. The heart and the metabolic organs and tissues were excised, rinsed, weighed, and counted.
Extraction of 99mTc(N)-DBODC(3/5) from Heart.
The excised heart was placed in saline (2 mL), chopped for 5 min, and centrifuged (3,000g for 10 min). Pellet and supernatant were counted for their radioactivity content; only 15% of the initial activity was found in the liquid fraction. The remaining activity was completely extracted from the tissue by treating the pellet with MeCN (2 mL) and chopping for 5 additional minutes. The organic solution was separated from the particulate by centrifugation (3,000g for 10 min). Aliquots of the aqueous and organic solutions, the latter previously diluted 1:3 with water, were eluted through an OASIS HLB extraction cartridge and analyzed by TLC and HPLC as described before.
Extraction of 99mTc(N)-DBODC(3/5) from Liver and Kidneys.
The extraction was performed as described above, and the eluate analyzed by TLC and HPLC.
Metabolites in Intestine.
The intestinal lumen was rinsed with 2 mL of saline; 90% of the total activity was collected in the endoluminal content. The liquid fraction was separated from the intestinal content by centrifugation (3,000g for 10 min) and counted. Fifty percent of the activity was found in this first liquid fraction. Exhaustive extraction of the residual activity from the solid fraction was performed by using MeCN (2 mL) followed by centrifugation (3,000g for 10 min). An aliquot of each fraction (1 mL) was eluted through an OASIS HLB extraction cartridge, and the eluate analyzed by TLC and HPLC.
Metabolites in Urine.
Urine was collected directly from the bladder and loaded onto an activated OASIS HLB extraction cartridge. The complex was eluted and analyzed by TLC and HPLC.
The method used to extract metabolites from tissues and fluids allowed for the recovery and analysis about 90% of the activity.
Effects of Cyclosporin A on Pharmacokinetic Properties of 99mTc(N)-DBODC(5).
Animals (n = 6) were treated with a solution of cyclosporin A (16 mg kg–1 in 250 µL of dimethyl sulfoxide intraperitoneally) 60 min before the radiotracer administration. At 30 and 120 min after injection, animals (n = 3) were sacrificed. As a blank, a group of animals (n = 6) received the carrier vehicle solution (dimethyl sulfoxide) by intraperitoneal injection 60 min before radiotracer administration.
Subcellular Distribution.
Subcellular distribution of 99mTc(N)-DBODC(5) was evaluated in ex vivo myocardial tissue, excised 15 min after injection, by applying a previously reported procedure (8). For comparison, the subcellular distributions of 99mTc-sestamibi and 99mTc-tetrofosmin were assessed in the same experiment.
99mTc-activity in each supernatant and pellet was measured in a -counter.
Malate dehydrogenase, a mitochondrial inner matrix enzyme, was used as a marker to determine the distribution and association of mitochondrial contents into various fractions (9).
CCCP experiments (final concentration, 5 µmol dm–1) were performed to define the accumulation of 99mTc-complexes in the intact mitochondria dispersed into each fraction. CCCP, in fact, uncouples the influx of protons from ATP synthesis, inducing a collapse of the mitochondrial membrane potential, resulting in release of activity from the mitochondrial pellet in the cytosolic fractions (supernatants) (8,10,11).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Measures of the log P and log K0' values of 99mTc-agents revealed that 99mTc(N)-DBODC(3/5) complexes were more lipophilic than 99mTc-sestamibi. Staring from the more lipophilic compound, the corresponding log P and log K0' values were as follows: 99mTc(N)-DBODC(5): log P = 1.61 ± 0.12, log K0' = 3.68 ± 0.05; 99mTc(N)-DBODC(3): log P = 1.18 ± 0.15, log K0' = 3.35 ± 0.22; 99mTc-sestamibi: log P = 0.87 ± 0.18, log K0' = 3.22 ± 0.08.
Because of the fast blood clearance and the low liver uptake followed by rapid washout (Table 1), 99mTc(N)-tracer stability was first estimated in vitro. Thus, binding to the serum proteins and biotransformation of the two agents in blood and liver tissues were assessed by incubating the purified compounds at 37°C in human and rat serum and in rat liver homogenates.
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Figure 2 shows HPLC analysis of 99mTc(N)-DBODC(3/5) incubated in human serum. Each analysis displays 2 profiles, one resulting from ultraviolet detection of the serum proteins and the other from radiometric detection of 99mTc(N)-complexes. No 99mTc-activity was coeluted with any of the protein fraction, and essentially all activity was eluted in a single peak as 99mTc(N)-DBODC(3/5) complex. These observations established that, under these conditions, there was no detectable binding to serum components.
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These results indicated that 99mTc(N)-DBODC(3) and 99mTc(N)-DBODC(5) simply were distributed from the blood flow as free cations or that an extremely labile conjugation between the serum proteins and the complex could be involved, such that the 99mTc(N)-DBODC(3/5) dissociated during the elution.
In vitro metabolism studies performed in human and rat serum, as well as in rat liver homogenates, revealed that the radiolabeled compounds remain intact for prolonged incubation at 37°C.
The quantitative organ biodistribution of 99mTc(N)-DBODC(3/5) is reported in Table 1.
Data show high and persistent myocardial uptake in both female and male rats. However, washout of 99mTc(N)-DBODC(5) from nontarget tissues, such as lungs, liver, and kidneys, was remarkably higher in female than in male rats; as a consequence, the heart-to-lung and heart-to-liver ratios were found to decrease in male rats.
The distribution of 99mTc(N)-DBODC(5) activity in principal organs with or without cyclosporin A administration is reported in Table 2 and Figure 3. The effects of cyclosporin A on the pharmacokinetic profile were evident at 30 and 120 min after injection. Notable reductions in lung, liver, and kidney washout (Figs. 3A and 3B) and significant variations in the activity distribution in the intestinal tract (Figs. 3C and 3D) were observed. No variation in the biodistribution profile of 99mTc(N)-DBODC(5) was observed after administration of a dimethyl sulfoxide by intraperitoneal injection 60 min before radiotracer.
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TLC and HPLC analysis was performed on the recovered aqueous and organic solutions to investigate the effect of the uptake process on the chemical identity of the complex. The chromatographic profiles shown in Figures 4B and 4C demonstrated that the myocardial uptake process and the corresponding trapping mechanism do not involve any change in the chemical nature of the tracer (control peak, Fig. 4A). Similarly, the activity extracted from other tissues and fluids in female rats at 30 and 120 min after injection evidenced that the radiotracer was stable in vivo.
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Subcellular distribution studies of 99mTc(N)-DBODC(5) were performed by standard differential centrifugation techniques using the commercially available compounds as a control (99mTc-sestamibi and 99mTc-tetrofosmin) (8). For all complexes, most activity was released in supernatants I and II (defined as the two cytosolic fractions isolated during the fractionation process), and only a small portion of the activity was found associated with the mitochondrial fraction (Fig. 5A).
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The corrected subcellular distribution of the complexes was estimated by correlating the distribution of the markers with the mitochondrial integrity. The result is reported in Figure 7.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Despite the presence of similar ether groups on their coordination sphere and the higher lipophilic character of both 99mTc(N)-DBODC(3/5) complexes, their pharmacokinetic properties were significantly different from those of 99mTc-sestamibi and 99mTc-tetrofosmin (3–5).
In particular, the excreted activity quickly moved from the hepatic tissue to the intestinal area, showing a high accumulation in this region also at 10 min after injection. The high intestinal accumulation may also account for the fast decrease of activity in the circulation and, consequently, in nontarget tissues, resulting in remarkably high heart-to-liver and heart-to-lung ratios.
The results presented here clearly show that metabolism of these 99mTc(N)-complexes cannot explain their pharmacokinetic properties (3–5). Specifically, experimental data appear to support an important role played by P-glycoproteins (Pgp) or multidrug resistance–associated protein (MDR)–Pgp on the rapid elimination of these lipophilic compounds from the tissues (14–17).
Protein-binding studies demonstrated a negligible association with serum proteins, in agreement with the extremely fast in vivo distribution of the complexes characterized by a rapid blood clearance.
Biodistribution studies performed on both female and male rats showed high and prolonged heart uptake. Differences between the observed values of heart uptake reported here in female rats and previous data in the literature (2,3,18) could be attributed to differences in the composition of the injected solution. In particular, in our work we used a saline solution containing less than 5% (v/v) ethanol whereas other groups used a phosphate buffer solution (0.1 mol dm–1; pH 7.4) containing 10% (v/v) ethanol (2,3) or a saline solution containing 15% (w/w) propylene glycol (18).
Subcellular distribution studies showed that 86.3% ± 7.4% of 99mTc(N)-DBODC(5) was localized in the mitochondrial fraction, analogously to what was previously observed for 99mTc-sestamibi and 99mTc-tetrofosmin and supporting the conclusion that heart uptake occurs through an identical mechanism (8,10,11). The presence of higher 99mTc(N)-activity in the cell fragments could be explained by the higher lipophilic character of 99mTc(N)-DBODC(5), compared with the commercial compounds.
Extraction of activity localized in the heart tissue after intravenous injection of the 99mTc(N)-complexes revealed that the myocardial uptake and trapping processes do not involve a change in the chemical nature of the tracer. Similarly, analysis of the identity of complexes extracted from urine samples and from the liver of female rats did not show any metabolic transformation. Sex-related differences were observed in the liver and in the endoluminal content of male rats, where a low amount of hydrophilic compounds was detected. This fact could be related to the expression of the hepatic CYP450 which is considerably higher in male than in female rats (12). Metabolic studies performed in vitro by incubating, at 37°C, the purified 99mTc(N)-compounds in human serum, rat serum, and rat liver homogenates confirmed the high metabolic inertness of these compounds. These results displayed an irrelevant contribution of CYP450 enzymes to the pharmacokinetics of these agents, suggesting the participation of Pgp or MDR-Pgp in the efflux mechanism in 99mTc(N)-DBODC(5) from nontarget tissues.
Some evidence supporting the involvement of Pgp transporters in the pharmacokinetic profiles of these tracers was collected from biodistribution studies performed on female and male rats. In fact, biodistribution data showed a significantly reduced elimination of 99mTc(N)-DBODC(5) from the excretory organs of male rats, as compared with female rats. This finding is consistent with higher expression of Pgp/MDR-Pgp proteins in female than in male rats (12,13).
To confirm the role of these transporters in efflux of 99mTc(N)-agents from nontarget tissues, the effect of an MDR modulator, cyclosporin A, on biodistribution of 99mTc(N)-DBODC(5) was investigated (19–22). When cyclosporin A was administered before intravenous injection of the tracer in female and male rats, 99mTc(N)-DBODC(5) uptake in lungs, liver, and kidneys was increased with respect to the baseline, and excretion from nontarget tissues was delayed, with a concomitant reduction of the corresponding target-to-nontarget ratios (Table 2). The most important differences were in the distribution of activity in the intestinal tract of female rats (Fig. 3), with a significant increase of activity in the intestinal tissue and a concomitant decrease in the endoluminal content. This fact strongly indicates that it is intestinal Pgps/MDR-Pgps, rather than metabolizing enzymes, that plays the most important role in the prehepatic elimination of this compound (14). It is interesting to note that, after cyclosporin A treatment, heart activity was 30% higher than in the baseline studies (23).
All these changes were consistent with an enhanced intracellular concentration of the tracer due to inhibition of the Pgp function in healthy tissue by cyclosporin A.
Considering these results, it is reasonable to believe that the MDR Pgp transport function of hepatocytes and, in particular, of intestinal cells is responsible for the extremely fast efflux of 99mTc(N)-complexes from these regions. The difference in the excretion kinetics of 99mTc(N)-DBODC(5) with respect to 99mTc-sestamibi and 99mTc-tetrofosmin—a difference that results in significantly higher heart-to-liver ratios early after injection—could be justified by assuming that these 99mTc(N)-tracers can be recognized more specifically by the MDR-Pgps than the other two monocationic compounds. Consequently, it is reasonable to expect that these new compounds can be much more effective for noninvasive imaging of Pgp functions in different tumors (24–26).
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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The potential selective recognition of these 99mTc(N)-myocardial tracers as MDR-Pgp substrates, combined with their mitochondrial accumulation, may extend their clinical application from cardiology to oncology such as in tumor and MDR imaging studies. On the basis of these results, 99mTc(N)-DBODC(5) could represent a first example for developing more specific and sensitive tumor markers.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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References |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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); male, baseline (
); and male, cyclosporin A (
).
