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Research and Development, GE Healthcare Bio-Sciences, Little Chalfont, Buckinghamshire, United Kingdom (D.E., M.B., R.L., G.F., D.J.B., V.G., C.C., A.O.); and Institution of Oncology, Radiology, and Clinical Immunology, Section of Radiology, Uppsala University Hospital, Uppsala, Sweden (D.E., H.A.)
(Received February 6, 2006; accepted April 5, 2006)
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Abstract |
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). Rapid diagnosis is essential because the mortality rate associated with untreated pulmonary embolism can be up to 30%. Timely intervention can reduce this rate to as low as 2% (O'Neill et al., 2004). NC100668 (Fig. 1A) is based on a linear peptide with homology to the N-terminal sequence of 
2-antiplasmin, a known factor XIIIa (FXIIIa) substrate (Lijnen et al., 1987). FXIIIa catalyzes the formation 
-(
-glutamyl)lysyl bonds that hold the fibrin clot together (Gorman and Folk, 1984).
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-emitting radioisotope used in diagnostic nuclear medicine. Usually, 99mTc is complexed to a ligand before administration. The precise distribution of 99mTc is then determined by the physicochemical properties of the 99mTc-ligand complex. The distribution of radioactivity within the body can be detected externally using a -camera. The pattern of radioactive uptake and retention in the acquired image is then used to help make a diagnosis. Because of the short radioactive half-life of 99mTc-labeled diagnostic radiopharmaceuticals, they are usually distributed to the point of use as freeze-dried kits ready for radiolabeling. Before use, kits are reconstituted with a saline solution containing 99mTcO –4 (pertechnetate ion) obtained from a 99Mo/99mTc generator. To ensure that the radiolabeling process achieves the highest radiochemical purity (RCP) possible, the ligand is present in vast excess in relation to the amount of 99mTcO –4 added. Typically, less than 1% of the ligand present is bound to 99mTc following reconstitution. The NC100668 kits are all formulated with 100 µg of ligand; therefore, in a reconstituted vial less than 1 µg is chelated to 99mTc.
Changes in the physicochemical properties (e.g., net charge, lipophilicity) of a molecule occur following chelation of 99mTc. These changes can affect the biological behavior of the molecule relative to its unchelated analog. For example, the dopamine transporter tracer TRODAT cannot cross the blood-brain barrier, whereas 99mTc-chelated TRODAT can (Kung et al., 1997). These changes may also alter the affinity and selectivity of a 99mTc-chelated ligand for metabolizing enzymes relative to its unchelated counterpart. Therefore, the radiolabeled metabolites present must be characterized independently of the unlabeled metabolites to ensure that any potential for accumulation and retention of radioactivity in an unrelated organ or pathology is understood. In the event that this information is not available, it may be possible that on some occasions a false-positive diagnosis might be made. Given the low chemical concentration of radiolabeled material involved, characterization of any radiolabeled metabolites present is not a trivial challenge and cannot be addressed by conventional liquid chromatography/mass spectrometry techniques alone.
The purpose of this study was to investigate the in vivo metabolism of 99mTc-NC100668 following i.v. injection to rats and to identify the major radioactive metabolites present by cochromatography with authentic potential metabolites. The in vitro stability of 99mTc-NC100668 was studied in whole blood and plasma, as well as in hepatic S9 (a liver homogenate-rich cytochrome P450) of rat origin, and compared with that obtained from samples of human origin. The identity of the major metabolite was confirmed by cochromatography with an authentic standard using a separate high-performance liquid chromatography (HPLC) method. Finally, the impact of the presence of the major 99mTc-metabolite on the clinical usefulness was studied by a combination of in vivo biodistribution and in vitro plasma clot uptake experiments.
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Materials and Methods |
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After cooling, the RCP of each preparation was determined by reverse-phase HPLC and silica gel instant thin layer chromatography (ITLC). HPLC (Gilson, Luton, UK) was performed by applying aliquots of each preparation to a 250 mm x 4.6 mm (5 µm) Luna C18(2) column (Phenomenex, Macclesfield, UK) that was eluted with 0.05% trifluoroacetic acid (TFA) (Sigma, Gillingham, UK) in water (solvent A) (VWR International, Poole, UK) and 0.04% TFA in acetonitrile (solvent B) (Rathburn, Walkerburn, UK). A linear gradient from 16% solvent B to 40% solvent B was applied over 20 min at a flow rate of 1 ml/min. 99mTc-labeled species were detected postcolumn using a Bioscan radiodetector (Lablogic, Sheffield, UK).
ITLC was carried out using ITLC-silica gel strips (Pall, Portsmouth, UK) run in a cylinder containing a 50:50 mixture (v/v) of methanol (VWR International) and 1 M ammonium acetate (VWR International). The strips were allowed to dry and were scanned on a Packard Instantimager (PerkinElmer, Beaconsfield, UK). The image produced was integrated around the origin to give the amount of reduced hydrolyzed technetium (RHT) (99mTc) in the preparation. All the other radiolabeled species moved with the solvent front.
All the 99mTc-NC100668 preparations had an RCP of greater than 85%. The major impurity of 99mTc-NC100668 was the kinetic 99mTc-complex of NC100668, which accounted for at least 6% of the radioactivity present. Under radiolabeling conditions, the kinetic 99mTc-complex of NC100668 was rapidly formed. Heat was used to convert most of this through to the thermodynamic complex, 99mTc-NC100668. The kinetic 99mTc-complex of NC100668 is believed to be a different arrangement of the technetium amine-oxime complex. The remaining radiochemical impurities were RHT (99mTc) (approximately 1%), 99mTc-methylene diphosphonic acid (MDP), and hydrolytic decomposition products of 99mTc-NC100668. The RCP of the 99mTc-NC100194 preparations used was at least 45 and 85% for the 99mTc-complex of Gly-NC100194 preparations used. The remaining radioactivity in these preparations was associated with 99mTc-MDP and 99mTcO –4.
99mTc-diethylenetriaminepentaacetic acid (DTPA) was prepared by addition of 5 ml of sodium pertechnetate (99mTc) solution, containing 1 GBq of 99mTc, to a commercially available labeling kit containing calcium trisodium diethylenetriaminepentaacetate (GE Healthcare). Each preparation was allowed to stand for 30 min before RCP analysis by silica gel ITLC according to the manufacturer's instructions. The 99mTc-DTPA preparations used in this study had an RCP of at least 94% before use. All the other chemicals were of analytical grade quality.
In Vitro Stability of 99mTc-NC100668. An aliquot of 99mTc-NC100668 (50 MBq) was added to glass scintillation vials containing 1.9 ml of prewarmed saline or plasma (obtained from male Wistar rats and human volunteers, anticoagulated with sodium citrate). 99mTc-NC100668 (75 MBq) was added to glass scintillation vials containing 3 ml of prewarmed citrate anticoagulated rat or human blood. Samples of plasma or blood were removed after 5, 45 (60 min for the saline samples), and 90 min of incubation at 37°C in a shaking water bath.
99mTc-NC100668 (25 MBq) was dispensed into prewarmed scintillation vials containing 3 ml of rat bile or urine. The vials were returned to the 37°C shaking water bath. Samples were removed after 5, 60, and 120 min of incubation. All the incubations were performed in duplicate.
In Vitro Metabolism of 99mTc-NC100668. Twenty microliters of a 10-mg/ml ß-NADPH (Sigma) solution and 10 µl of a 20-mg/ml protein S9 fraction (Xenotech, Lenexa, KS) obtained from either rat or human liver was added to 1.69 ml of 50 mM tris(hydroxymethyl)hydrogen chloride buffer (Sigma), pH adjusted to 7.4, and prewarmed to 37°C. 99mTc-NC100668 (10 MBq) was then added. The hepatic S9 mixture was placed in a shaking 37°C water bath. Samples were removed immediately after the addition of 99mTc-NC100668 (defined as 0 min for the purposes of this experiment) and after 45 and 90 min of incubation. All the incubations were performed in duplicate and analyzed separately.
Cytochrome P450-independent metabolism was investigated by incubating 99mTc-NC100668 in rat and human hepatic S9 fractions deficient in ß-NADPH over 90 min at 37°C.
Hydrolysis 99mTc-NC100668 by Trypsin. Immobilized trypsin beads (200 units/ml) (Perbio Science, Cramlington, UK) were prepared for use by washing three times in 1 ml of 20 mM calcium chloride solution (Sigma). After each wash, the trypsin bead pellet was collected by centrifugation at 10,000 rpm in a bench-top centrifuge for 10 min. After the third wash, the trypsin bead pellet was resuspended in 1 ml of 20 mM calcium chloride. A 50-µl aliquot (containing approximately 10 units of enzyme activity) was removed and added to 0.85 ml of 20 mM calcium chloride, to which was added 99mTc-NC100668 (50 MBq). The mixture was incubated for 60 min at 37°C before analysis.
In Vivo Metabolism of 99mTc-NC100668. Six male Wistar outbred rats [Crl: (WI) BR] (Charles River, Margate, UK) weighing between 180 and 260 g at the time of study were injected with 99mTc-NC100668 (50 MBq) via the lateral tail vein under light anesthesia with halothane (Merial, Harlow, UK) (6% in O2). The animals were then deeply anesthetized following the administration of urethane (150 mg/kg) (Sigma) by i.p. injection. This level of anesthesia was maintained by further i.p. injection of urethane as required. Three animals were killed by cervical dislocation 5 min after injection, and three animals were killed 60 min after injection.
At the time of sacrifice, the abdomen of each animal was opened and the viscera moved to one side to expose the bladder and vena cava. A venous blood sample of 3 ml was taken from the vena cava using a 10-ml syringe fitted with a 25-gauge hypodermic needle containing sodium citrate solution. After the blood sample had been collected, a urine sample was taken by aspiration of the bladder using a separate needle and syringe.
A third group of three adult male Wistar rats was lightly sedated with halothane (6% in O2) and then deeply anesthetized by an i.p. injection of urethane. The right femoral vein was exposed and cannulated with a 25-gauge needle attached to a 5-cm length of tubing, with an internal diameter of 0.4 mm and external diameter of 1.0 mm (Portex, Hythe, UK).
A portion of the bile duct approximately 0.5 cm from the liver was exposed after laparotomy. This was then cannulated using tubing with an internal diameter of 0.5 mm and an external diameter of 1.0 mm (Portex). The viscera of each animal were laid to one side and wrapped in damp gauze. For the duration of the study, the gauze was kept moist by the regular application of 0.9% saline (Fresenius, Warrington, UK). Bile was allowed to flow for 5 to 10 min to ensure the patency of the cannula.
After the preparation period, 99mTc-NC100668 (50 MBq) was administered via the femoral cannula. Bile samples were collected through the bile duct cannula over three collection periods: 0 to 15 min, 16 to 45 min, and 46 to 90 min after injection.
Preparation of 99mTc-Labeled Samples for HPLC Analysis. Samples of blood were centrifuged for 20 min to remove the blood cells. Once the red blood cells had been removed, the blood samples were treated as plasma samples. The plasma and hepatic S9 samples were added to ice-cold acetonitrile. A 200-µl sample was added to 800 µl of acetonitrile. This was followed by centrifugation at 10,000 rpm with a fixed angle bench-top centrifuge for 10 min to sediment precipitated protein. The samples of bile and urine were collected and centrifuged in a bench-top centrifuge for 5 min at 10,000 rpm before analysis by HPLC. The supernatants were separated from the precipitated material, 100 µl of which was diluted with an appropriate volume of deionized water such that approximately 50 KBq of radioactivity could immediately be analyzed by HPLC with an on-line radiochemical detector (PerkinElmer).
HPLC Analysis of 99mTc-NC100668 Metabolites. HPLC was used to analyze the radiochemical profile of the metabolites of 99mTc-NC100668. In the initial studies, radiolabeled species were separated on a VYDAC C18 (250 mm x 4.6 mm, 5 µm) HPLC column (Hichrom, Reading, UK). A C18 Nova-Pak guard column (Waters, Watford, UK) was fitted in front of the HPLC column. The HPLC column was eluted at a constant flow rate of 1 ml/min. The separation was performed at ambient temperature. The aqueous mobile phase (solvent A) was 0.1% (v/v) TFA in analytical grade water, and the organic mobile phase (solvent B) was 0.1% (v/v) TFA in acetonitrile. The gradient used to elute the radiolabeled metabolites is described in Table 1.
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Cochromatography studies were performed to identify the principal metabolite present. In these experiments, processed urine samples containing the major metabolite were combined with either 99mTc-NC100194 or 99mTc-complex of Gly-NC100194 (produced by the trypsin-mediated proteolysis of 99mTc-NC100668). The amounts of radioactivity in the 99mTc-NC100194 and 99mTc-complex of Gly-NC100194 samples were diluted with analytical grade water until they were approximately equivalent to that in the processed urine sample. The resulting chromatograms were inspected to see whether peaks caused by the genuine metabolite and peaks caused by the presence of either 99mTc-NC100194 or 99mTc-complex of Gly-NC100194 could be resolved from one another.
A second HPLC method (referred to as the short HPLC method) was developed to confirm that the major metabolite present in urine and as a consequence of the trypsin-mediated cleavage of 99mTc-NC100668 would still coelute with the authentic standard of 99mTc-complex of Gly-NC100194. This method used a 250 x 4.6 mm (5 µm) Luna C18(2) column, which was eluted with 0.05% TFA in water (solvent A) and 0.05% TFA in acetonitrile (solvent B). A linear gradient from 5% solvent B to 10% solvent B was applied over 10 min at a flow rate of 1 ml/min. 99mTc-labeled species were detected post-column using a Bioscan radiodetector.
Biogeneration of 99mTc-Complex of Gly-NC100194. Eight male Wistar rats were lightly anesthetized with halothane (6% in O2) and injected i.p. with 1 part ketamine (100 mg/ml) (Pharmacia Animal Health, Corby, UK) and 1 part xylazine (2% w/v) (Bayer, Newbury, UK) at a dose of 1.0 ml/kg body weight. The right femoral vein of each animal was exposed. A cannula was inserted into the vein, and 0.5 ml of 99mTc-NC100668 (approximately 500 MBq) was injected. Once the 99mTc-NC100668 had been administered, the urethra of these animals was clamped to prevent loss of urine.
Sixty minutes after the administration of 99mTc-NC100668, each animal was sacrificed by cervical dislocation. The abdomen was opened, and the urine removed from the bladder. The urine from the eight animals was pooled. The radioactive content of the pooled urine was determined using an Ion Chamber (Capintec, Ramsey, NJ), and the radiochemical composition was determined by HPLC. The RCP of 99mTc-complex of Gly-NC100194 prepared in this way was determined to be more than 95% by HPLC.
Biodistribution of 99mTc-Complex of Gly-NC100194. Male Wistar out-bred rats [Crl: (WI) BR] weighing between 180 and 260 g were injected with a bolus of the biogenerated 99mTc-complex of Gly-NC100194 (50 MBq) via a lateral tail vein under light anesthesia with halothane (6% in O2). After injection, animals were individually housed in metabolism cages to allow separate collection of voided urine and feces. Three males were sacrificed at 2, 10, and 20 min, and three were killed 1, 4, and 7 h after injection.
Animals were killed by cervical dislocation under halothane anesthesia. Each carcass was weighed, and after dissection the percentage injected dose (% id) in the tissues and organs was determined by assay for radioactivity in a twin crystal automatic gamma counter. Blood, bone, muscle, skin, and fat samples were collected into preweighed containers and assayed for radioactivity; appropriate body composition factors (blood 5.8% b.wt., bone 5% b.wt., muscle 43% b.wt., skin 18% b.wt., and fat 7% b.wt.) were applied to data for these tissue samples to calculate the % id in the entire tissue. All the other tissues, organs, and excreta as listed in Table 3 were collected intact at dissection, and the % id was calculated as a percentage of the total radioactive dose administered.
In Vitro Plasma Clot Assay. 99mTc-complex of Gly-NC100194, 99mTc-NC100668, and 99mTc-DTPA were each added to separate plasma samples obtained from rat, which were anticoagulated with 100 mM sodium citrate (10% v/v) (Sigma). Aliquots of 0.4 ml of plasma were then added to P7 vials (Patterson Scientific, Luton, UK) containing either 0.84 ml of 110 mM calcium chloride (Sigma) with 40 units of bovine thrombin (Sigma) in a 50 mM tris(hydroxymethyl)aminomethane buffer (Sigma) (the plasma clot-forming buffer) or 0.84 ml of 50 mM tris(hydroxymethyl)aminomethane buffer only (the non-clot-forming buffer). Equivalent volumes of plasma, each spiked with an identical amount of each test substance, were also dispensed into P7 vials to act as counting standards.
After incubation of both the clot-forming and non-clot-forming mixtures at ambient temperature for 1 h, the clotting reaction was terminated by the addition of 1 ml of 0.4 M EDTA (VWR International). Each vial was washed with 50 mM tris(hydroxymethyl)aminomethane buffer containing 0.1% (v/v) Tween 20 detergent (Sigma), and the vial contents were dried on nitrocellulose filters with a pore diameter of 0.45 µm (Whatman, Maidstone, UK) over a vacuum manifold (Millipore, Watford, UK). Before use, these filters were pretreated by incubating them with 1.5% (w/v) bovine serum albumin (Sigma) overnight in a refrigerator at 2 to 8°C to minimize nonspecific binding.
The radioactivity present on these dried filters and in plasma counting standards was then measured using a sodium iodide gamma scintillation counter (PerkinElmer).
The plasma clot uptake (resulting from either specific or nonspecific incorporation) was expressed as a percentage of the total amount of each test substance added to each vial after subtraction of nonspecific binding to the filter observed in the non-clot-forming buffer.
Analysis of Chromatographic Data. The chromatographic data obtained using the radiochemical detector was processed using either Gilson Unipoint version 2.1 or Dionex Chromeleon version 6 software.
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In contrast, there was much less evidence of any metabolism occurring in human blood or plasma. After 90-min incubation, the amount of radioactivity associated with 99mTc-NC100668 decreased by less than 3 and 1%, respectively (data not shown).
In Vitro Metabolism of 99mTc-NC100668. The rate of turnover of 99mTc-NC100668 was faster in rat than in humans (Fig. 3); after 90-min incubation, less than 4% of the test substance remained in rat hepatic S9 compared with 52% in human hepatic S9. The major metabolite present in rat hepatic S9 fraction after 90 min was identified as 99mTc-complex of Gly-NC100194 (retention time, approximately 10 min). Three additional metabolites were observed in human and rat hepatic S9, with retention times of between 11 and 19 min. With the exception of the metabolite that eluted after approximately 15 min, the remaining species were all the minor components of the matrix; individually, each species represented no more than 2% of the radioactivity present. None of these species were observed in vivo; they most likely arise because of the partial proteolytic cleavage of the test substance. The metabolic profile produced when 99mTc-NC100668 was incubated with hepatic S9 in the absence of ß-NADPH was identical to that seen in the presence of ß-NADPH.
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When authentic 99mTc-NC100194 was cochromatographed with 99mTc-NC100668 that had been treated with trypsin, it was observed that the trypsin metabolite and 99mTc-NC100194 species could still be distinguished from each other.
Confirmation of the Identity of the Major Metabolites of 99mTc-NC100668 Using the Short HPLC Method. It was possible to resolve the authentic standards (99mTc-complex of Gly-NC100194 and 99mTc-NC100194) when a mixture of the two was analyzed using the short HPLC method (Fig. 5). 99mTc-NC100194 had a retention time of approximately 7 min compared with 7.5 min for 99mTc-complex of Gly-NC100194. The peaks that eluted before the two standards corresponded to 99mTcO –4 and 99mTc-MDP left over from the radiolabeling reaction.
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Using this HPLC method, it was possible to confirm that the major metabolite present after the hydrolysis of 99mTc-NC100668 in the presence of trypsin coresolved with authentic 99mTc-complex of Gly-NC100194 standard and not 99mTc-NC100194 (Fig. 6, A–C). Similarly, the presence of 99mTc-complex of Gly-NC100194 and 99mTc-NC100194 as the two metabolites present in urine was confirmed by cochromatography using authentic standards on the same system (Fig. 6, D–F).
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99mTc-NC100668 was stable in bile and urine. During 90 min of incubation, very little degradation of the test substance was observed. The only species observed to grow over time was 99mTcO –4.
Biodistribution of 99mTc-Complex of Gly-NC100194 in Rats. The clearance of 99mTc-complex of Gly-NC100194 from blood was rapid. Only 18% id was retained in the blood by 2 min, and after 7 h less than 0.1% id was present (Table 2). There was some uptake of radioactivity into lung (2% id) and heart (1% id) 2 min after injection. Radioactivity was rapidly lost from these two tissues. Twenty minutes after injection, the % id retained in these tissues had more than halved.
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The distribution of 99mTc-complex of Gly-NC100194 to other organs and tissues was rapid, with a high % id found in muscle (26% id), skin (21% id), and kidneys (12% id) 2 min after injection. By 1 h after injection, the % id retained within muscle (2% id), skin (3% id), and kidneys (4% id) had significantly declined. By 7 h after injection, only the kidneys (3% id) and liver (4% id) still retained more than 1% id. After 2 min, 4% id was detected in the liver. This increased to 6% id by 1 h, before returning to 4% by 7 h after injection.
In Vitro Plasma Clot Assay. The percentage uptake of 99mTc-complex of Gly-NC100194 (3 ± 1%) into the rat-forming clot assay was far less than that for 99mTc-NC100668 (43 ± 1%) and comparable with the value obtained for 99mTc-DTPA, the negative control (2 ± 1%).
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The identity of the major metabolite, the 99mTc-complex of Gly-NC100194, was determined from the findings that this metabolite had a retention time similar, but not identical, to that of 99mTc-NC100194, suggesting that the two molecules have similar structures. The plasma clot binding assay indicated that there were no groups available to this metabolite to permit FXIIIa-mediated incorporation into the forming plasma clot. This fact suggested that the lysine residue at position 12 was absent, as other small molecules with free NH2 groups (e.g., dansylcadaverine) have shown themselves to be effective FXIIIa substrates (Hantagan, 1982). The protease trypsin cleaves to the C terminus of basic amino acids; in the case of 99mTc-NC100668, a potential cleavage site exists after the lysine residue at position 12. After hydrolysis of 99mTc-NC100668 with trypsin in vitro, only a single radiolabeled species was observed, which did not coresolve with 99mTc-NC100194. By implication, the most likely identity of this metabolite is the 99mTc-complex of Gly-NC100194. Finally, it has been shown that the major urinary metabolite of the NC100668 ligand is Gly-NC100194 (Skotland et al., 2006). The identity of the major metabolite was then confirmed based on the cochromatography with an authentic 99mTc-complex of Gly-NC100194 standard using a second HPLC method (the short method).
Given the difference in charge and lipophilicity between NC100668 and 99mTc-NC100668, which would occur as a result of 99mTc chelation, it was possible that the metabolism of 99mTc-NC100668 might not be identical to that of NC100668. If the radiolabeled metabolites were not characterized, then there was always the possibility that they could be retained in an unrelated pathology, with the consequence for a false-positive diagnosis being made. In this case, it appears from the similarity of the metabolic profile of 99mTc-NC100668 and NC100668 that both entities are metabolized by the same trypsin-like proteases that are abundant in blood and kidney brush boarder (Skotland et al., 2006).
In addition, small amounts of 99mTc-NC100194 were observed in urine and bile after the administration of 99mTc-NC100668 to rat. After i.v. administration of NC100668, small amounts of NC100194 were also observed in urine (Skotland et al., 2006). The occurrence of NC100194 was ascribed to the metabolism of Gly-NC100194 by alanyl aminopeptidase (EC.3.4.11.2) in the kidneys. The presence of 99mTc-NC100194 would suggest that 99mTc-complex of Gly-NC100194 is also a substrate for this enzyme.
The biodistribution of the 99mTc-complex of Gly-NC100194 in rat indicates that it was rapidly removed from the body, principally by urinary excretion. The values quoted in this study are in fact slightly exaggerated because the biodistribution data are not corrected for the residual radioactivity in the blood contained within the tissues. Therefore, the actual amount of radioactivity associated with each of these organs will be slightly lower than stated here.
The disposition of 99mTcO –4 has been studied previously (McElvany et al., 1981); following administration, 99mTcO –4 is widely distributed throughout the body with elimination predominantly via the kidneys (although a significant hepatobiliary route of elimination also exists). Retention of radioactivity is noted in the gastric mucosa, choroid plexus, thyroid, and salivary glands.
In comparison, the distribution and elimination of 99mTc-NC100668 are rapid (Edwards et al., 2006). Following i.v. administration to rat, 99mTc-NC100668 is rapidly removed from all the tissues apart from liver and kidneys (3% id and 17% id at 1 h after injection, respectively), with elimination primarily into the urine (66% id at 1 h after injection).
The biodistribution data indicate that the presence of either the 99mTc-complex of Gly-NC100194 or TcO –4 in vivo is unlikely to adversely affect the ability of 99mTc-NC100668 to image blood clots. The rapid clearance of 99mTc-complex of Gly-NC100194 with no obvious sites of retention indicates that its presence is unlikely to give rise to a false-positive diagnosis or an adverse impact on the radiation dosimetry.
A number of other minor radiolabeled species were observed in vitro; most likely, they are the product of protease activity and chemical degradation of 99mTc-NC100668. These species were only observed after prolonged incubation and are not physiologically relevant given the rapid elimination of 99mTc-NC100668 in vivo. In vitro in bile, urine, and saline, the only species observed to grow over time was 99mTcO –4, this being most likely a consequence of radiochemical degradation.
The RCP of the preparations used in this study were at least 85%. However, inspection of Fig. 2 suggests that RCP was in fact higher. The major impurities of the 99mTc-NC100668 labeling reaction, the kinetic 99mTc-complex of NC100668 and 99mTc-MDP, are in fact intermediates of the final 99mTc-labeled product. The kinetic 99mTc-complex of NC100668 is rapidly converted to the final thermodynamic product on heating. The kinetic 99mTc-complex of NC100668 and the final product are believed to be different arrangements of the technetium-oxime complex. 99mTc-MDP is present because MDP is incorporated into the kit as Sn2+ solubilizer (Sn2+ is present to reduce the Tc ion to the correct oxidation state to permit chelation to NC100668). MDP also stabilizes the reduced Tc atom long enough to allow incorporation into the NC100668 molecule. Most likely, the act of adding the test compound to prewarmed incubation mixture drives the labeling reaction to completion. As a result initially, the RCP appears higher than was originally stated. The only significant remaining impurity, RHT (99mTc), is a colloid, and as a result any of this material present will be trapped at the head of the HPLC column and will not show up on an HPLC trace.
There are reports in the literature of mass spectrometry being used to elucidate the structures of radiopharmaceuticals (Vanbilloen et al., 2003; Vanderghinste et al., 2003). Despite this, it has yet to be shown that it is possible to identify the structures of the radiolabeled metabolites using such technology. This is because of the low chemical concentration of radiopharmaceutical present after administration to the test system. In addition, there are potential problems associated with the extraction and concentration of short-lived gamma-emitting isotopes (radioactive decay to other elements and radiolytic decomposition). Until these issues can be satisfactorily resolved, other methods, such as cochromatographic identification with authentic standards, must be used to identify the radioactive metabolites associated with novel radiotracers like 99mTc-NC100668.
In conclusion, this study has identified that the major radiolabeled metabolite of 99mTc-NC100668 in vivo in rat is the 99mTc-complex of Gly-NC100194. This metabolite is also present in human hepatic S9 after incubation with 99mTc-NC100668, suggesting that this metabolite would also be present in humans after administration of 99mTc-NC100668. Biodistribution and plasma clot uptake studies have shown that the 99mTc-complex of Gly-NC100194 will not have a negative impact on the clinical utility of 99mTc-NC100668.
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Acknowledgments |
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ABBREVIATIONS: NC100668, Acetyl-Asn-Gln-Glu-Gln-Val-Ser-Pro-Tyr(3-iodo)-Thr-Leu-Leu-Lys-Gly-NC100194; FXIIIa, factor XIIIa; RCP, radiochemical purity; TRODAT, ethanethiol, 2-[[2-[[[(1R,2R,3S,5S)-3-(4-chlorophenyl)-8-methyl-8-azabicyclo[3.2.1]oct-2-yl]methyl](2-mercaptoethyl)amino]ethyl]amino]; HPLC, high-performance liquid chromatography; NC100194, N,N-Bis(N-(1,1-dimethyl-2-(hydroxylimino)propyl)aminoethyl)aminoethylamine; ITLC, instant thin layer chromatography; TFA, trifluoroacetic acid; RHT, reduced hydrolyzed technetium; MDP, methylene diphosphonic acid; DTPA, diethylenetriaminepentaacetic acid; % id, percentage of injected dose.
Address correspondence to: David Edwards, Research and Development, GE Healthcare Bio-Sciences, The Grove Centre, Little Chalfont, Buckinghamshire, HP7 9LL, UK. E-mail: davidedwards{at}ge.com
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  K. G. Toft, J. A. Johnson, I. Oulie, and T. Skotland NC100668, a New Tracer Tested for Imaging of Venous Thromboembolism: Pharmacokinetics and Metabolism in Humans Drug Metab. Dispos., November 1, 2007; 35(11): 1979 - 1984. [Abstract] [Full Text] [PDF]
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