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This Article Abstract Full Text (PDF) All Versions of this Article: dmd.106.014126v1 35/11/1979    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Toft, K. G. Articles by Skotland, T. Search for Related Content PubMed PubMed Citation Articles by Toft, K. G. Articles by Skotland, T.

SHORT COMMUNICATION

NC100668, a New Tracer Tested for Imaging of Venous Thromboembolism: Pharmacokinetics and Metabolism in Humans

Research and Development, GE Healthcare, Oslo, Norway (K.G.T., I.O., T.S.) and Princeton, New Jersey (J.A.J.)

(Received January 3, 2007; accepted August 2, 2007)

	Abstract

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The ^99mTc-complex of NC100668 [Acetyl-Asn-Gln-Glu-Gln-Val-Ser-Pro-Tyr(3-iodo)-Thr-Leu-Leu-Lys-Gly-NC100194] is a new tracer tested for nuclear medical imaging of venous thromboembolism. NC100668 is a 13-amino acid peptide with a Tc-binding chelator [NC100194; -NH-CH₂-CH₂-N(CH₂-CH₂-NH-C(CH₃)₂-C(CH₃)=N-OH)₂] linked to the C-terminal end. The present study was performed following injection of ^99mTc-NC100668 in healthy human volunteers with five dose levels of NC100668 (20-2000 µg) and a constant radioactivity dose. The rate at which the radioactivity was cleared from blood was independent of gender and dose of NC100668; more than half of the 82% urinary clearance of radioactivity was obtained 2 h postinjection. The radioactivity in blood was reduced to 50% of initial values within 12 min; this was followed by a more gradual decrease with a half-life of 1.2 h and a terminal elimination half-life of 10.5 h. The plasma concentration of NC100668 decreased rapidly with an initial half-life of 5 to 10 min. The half-life after this initial phase could be estimated for only two of the subjects in the highest-dose group because the NC100668 concentration in the other samples at these time points was below the limit of detection of the liquid chromatography/mass spectrometry (LC/MS) method. LC/MS analyses of urine samples revealed the identity of two metabolites generated from the C-terminal end of the molecule; Gly-NC100194 was identified as the major metabolite and NC100194 as a minor metabolite. The estimated sum of these two metabolites is in the same magnitude as the recoveries of ^99mTc in these samples, indicating that most of the ^99mTc excreted in urine is bound to one of these metabolites.

Diagnostic radiopharmaceuticals are radioactive substances used for medical imaging of a variety of diseases. ^99mTc (t of 6.02 h) is the -emitter most often used in these agents (Liu et al., 1997). The ^99mTc-based agents are distributed to hospitals in a Tc-free form as a freeze-dried product ready for labeling with technetium, which is eluted from a ⁹⁹Mo/^99mTc generator and added to the product on the day of imaging. These radiopharmaceuticals contain a vast excess of the chelating agent compared with the added Tc; typically less than 1% of the injected agent is in the form of the Tc complex (sum of the -emitter ^99mTc and the non--emitter ⁹⁹Tc, formed from ^99mTc). Hence, the unlabeled chelating agent makes up almost the entire amount of the chemical entity injected, whereas it is the very small amount of the ^99mTc-labeled agent that is responsible for the imaging effect.

We have earlier described a rapid clearance from blood of NC100668 following injection in rats, and that two metabolites and no parent compound were identified in rat urine (Skotland et al., 2006). The present data were obtained using samples collected in a Phase I study performed as a placebo-controlled, observer-blinded, randomized, single ascending dose study with five i.v. dose levels of ^99mTc-NC100668 injected into healthy volunteers. The injected radioactivity was kept constant at 190 MBq per subject, whereas five dose levels of NC100668 (20-2000 µg) were injected (imaging dose expected to be 100 µg).

	Materials and Methods

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Reagents and Substances. All the chemicals and substances used were as previously described in the rat metabolism study (Skotland et al., 2006).

In Life Part of the Study and Sample Collection. ^99mTc-NC100668 was prepared using sterile conditions and aseptic techniques by reconstitution of the NC100668 Kits with Sodium (^99mTc) Pertechnetate Injection USP/Ph.Eur., followed by heating at 60 ± 5°C for 10 min. The reconstituted ^99mTc-NC100668 was used within 2 h and was not to be administered if a high-performance liquid chromatography method showed the ^99mTc-NC100668 peak to be less than 85% of the total radioactivity or any other peak to be more than 7% of total, or if an instant thin-layer chromatography method showed reduced hydrolyzed technetium of more than 5% of total.

The study included eight subjects receiving ^99mTc-NC100668 for each of the five dose levels (a total of 40 subjects). The nominal dose of NC100668 was 20, 100, 500, 1000, and 2000 µg with a constant radioactivity of 187 ± 12 MBq (mean ± S.D.). The molar ratio of ^99mTc-NC100668 to NC100668 is approximately 5% at the lowest dose, 0.05% at the highest dose, and close to 1% at the expected clinical dose of 100 µg. The age of the subjects ranged from 19 to 64 years (mean age at each dose level, 31-47 years). There were more males than females; no females in the lowest-dose group, three females in the highest-dose group, and two females in the other dose groups. The study was conducted in full accordance with the current version of the Declaration of Helsinki.

View larger version (18K): [in this window] [in a new window]   FIG. 1. Mean plasma concentration of radioactivity (A) and mean plasma concentration with S.D. of NC100668 (B) following administration of 99mTc-NC100668 injection. The nominal dose of radioactivity was 190 MBq for all the subjects, whereas the nominal doses of NC100668 were 2000 µg(), 1000 µg(), 500 µg (), 100 µg(), and 20 µg().

Quantification of NC100668 in Blood Samples. The concentration of NC100668 was analyzed using a validated LC/MS method (Toft et al., 2005).

Metabolite Identification in Urine Samples Using LC/MS Analysis. Just before LC/MS analysis the samples were thawed, centrifuged at 3000g for 10 min, and cleaned by a solid-phase extraction (SPE) procedure using C18-SPE columns (100 mg, 1 ml from Varian Inc., Palo Alto, CA). These columns were conditioned with 2 x 1 ml of methanol and equilibrated with 2 x 1mlof water. The urine samples (100 µl) were then applied onto the SPE cartridges, which thereafter were washed with 2 x 1 ml of water. The cartridges were then eluted with 2 x 0.5 ml of 40% methanol in HCl into plastic cryotubes. Finally, the samples were evaporated under nitrogen and dissolved in 100 µl of sample solvent, mixed, and transferred to plastic micro high-performance liquid chromatography vials. Metabolite identification was performed using the LC/MS system previously described in the rat metabolism study (Skotland et al., 2006).

Estimation of Gly-NC100194 and NC100194 in Human Urine. The amounts of NC100194 and Gly-NC100194 in the urine samples were determined using the same LC/MS system used for metabolite identification by correlating the peak areas of the urine samples with the areas found for calibration standards. Calibration curves were made by plotting the peak area of NC100194 (m/z 345.3 ± 1.0) or Gly-NC100194 (m/z 402.3 ± 1.0) against the theoretical concentration of NC100194 or Gly-NC100194 in the calibration standards; the calibration curves were fitted to a nonlinear equation, y = a + bx + cx², and weighted with a weighing factor of 1/y².

Gly-NC100194 was estimated by comparing the signals with that obtained using calibration curves in the range 3.2 to 2000 ng/ml. The signal-to-noise ratio was approximately 4 for the lowest calibration sample. Although the goodness of fit values for the calibration points were within ± 15% (back-calculated values), the intermediate precision was rather poor, in the range of 30 to 60% for three control samples containing approximately 80, 280, and 1400 ng of Gly-NC100194/ml. It is believed that leakage from the SPE column made a major contribution to this poor precision.

NC100194 was estimated using calibration curves in the range 8 to 5000 ng/ml. The signal-to-noise ratio was approximately 4 for the lowest calibration sample. The validation data for this analysis were in the same range as that described above for Gly-NC100194.

Radiochemical Quantification of Blood and Urine Samples. The total radioactivity was determined using a Packard Cobra model 5003 gamma counter (PerkinElmer Life and Analytical Sciences, Boston, MA). Duplicate aliquots were assayed of each sample. The limit of quantification (LOQ) was defined as twice the background. All the gamma-counting data were decay-corrected to the time of injection and are presented in units of kilobecquerel per milliliter.

Analysis of Data and Pharmacokinetic Calculations. Analysis of chromatographic and mass spectrometry data was performed as previously described in the rat metabolism study (Skotland et al., 2006). Model-independent pharmacokinetic analyses were performed using PK Solutions 2.0 software (Summit Research Services, Montrose, CO). For pharmacokinetic calculations, dose was taken to be the nominal NC100668 dose administered.

	Results and Discussion

Top Abstract Materials and Methods Results and Discussion References

 
Pharmacokinetics of ^99mTc in Plasma. The mean plasma concentrations of radioactivity for all five doses of NC100668 are shown in Fig. 1A. The rate of clearance of radioactivity from plasma was independent of gender (data not shown). The C_max (range 14.8-18.6 kBq/ml for the five dose levels) and area under the curve (range 30.9-39.9 kBq · h/ml) for plasma blood activity were independent of the NC100668 dose over the 100-fold range studied. Of the 39 subjects evaluable for pharmacokinetic analysis, the individual concentration versus time profiles of seven subjects (18%) exhibited a biexponential decrease, whereas 32 (82%) exhibited a triexponential decrease. The initial decrease in radioactivity (generally estimated over the interval from 5 min to 30 or 60 min) was rapid and reduced to 50% of the observed initial values within approximately 12 min. The decline from 1 to 6 h postdose was more gradual with a half-life of approximately 1.2 h (range 1.18-1.31 h). Lastly, the observed elimination half-life estimated over the interval from 8 to 24 h was approximately 10.5 h (range 9.2-11.9 h). Clearance was estimated to 68 ml/h/kg (range 61.7-74.8 ml/h/kg) and the volume of distribution to 1040 ml/kg (range 1005-1084 ml/kg). Because the majority of the radioactivity (82%) was rapidly eliminated from the body via renal excretion during the distribution phase, the terminal elimination half-life should be interpreted with caution because this represents the disposition of a relatively minor fraction of the total administered dose. Therefore, the half-life for the second phase, which characterizes the elimination of the majority of the administered dose, is likely to be more clinically relevant and is consistent with removal via glomerular filtration.

Pharmacokinetics of ^99mTc in Urine. The renal clearance of radioactivity appeared to be independent of the dose of NC100668 and was estimated to be 57 ml/h/kg, which is approximately 83% of the estimated value of 68 ml/h/kg for the systemic clearance. The urinary recoveries were estimated to be 76 ± 17%, 81 ± 7%, 91 ± 15%, 89 ± 13%, and 72 ± 14% (percentage of injected dose; mean ± S.D.; from the lowest to the highest-dose group). Thus, approximately 82% of the injected radioactivity cleared through the kidneys, and more than half of this within 2 h postinjection.

View larger version (18K): [in this window] [in a new window]   FIG. 2. MS spectra (mass range 150-800) of human urine samples from subject 44; predose (A) and following injection of 1.6 mg of NC100668 (B). MS spectra were collected at the retention time of 11.60 to 12.00 min from the total ion current chromatograms (mass range 150-2000) shown in the insets.

The NC100668 plasma concentrations decreased rapidly with an initial half-life of 5 to 10 min. The elimination half-life could be estimated only for two subjects in the highest-dose group because the concentrations at later time points were below the LOQ. Consequently, the area under the curve values and clearance and volume of distribution could not be accurately assessed from these data.

When comparing data in Fig. 1, A and B, it should be noted that counting of radioactivity means counting the total radioactivity, not only ^99mTc-NC100668 but also any ^99mTc-containing impurities (such as the pertechnetate ion, ^99mTcO₄^-) and ^99mTc-NC100668 metabolites, and that only a very small fraction of NC100668 has ^99mTc bound to its C-terminal chelate, even at the lowest dose of NC100668. A more rapid elimination of NC100668 than the radioactivity indicated that metabolites of NC100668 were formed in blood, similar to that observed in rats (Skotland et al., 2006). However, too few data are available in the present study to conclude whether such metabolites were formed also in human blood.

Identification of Metabolites in Urine. The first part of the metabolite identification focused on analyzing the urine sample collected during the first hour after injection of NC100668 to subject 44 because this sample contained the highest metabolite concentration. Using the LC/MS method developed for analysis of urine metabolites following injection of high doses (up to 5 mg of NC100668/kg) in rats (Skotland et al., 2006), no difference was observed in the total ion chromatogram of the postdose urine sample when comparing with the predose sample (Fig. 2, A and B, insets), even in the region with retention time of 11.60 to 12.00 min, where differences were observed in the rat study. However, as also shown in Fig. 2, A and B, differences in signal intensities were observed in the MS spectrum in this region of the chromatogram in the postdose sample; new signals were observed at m/z 201.6 and 402.2, and new, but much weaker, signals were observed at m/z 303.3 and 345.2.

The substance in the human postdose urine sample giving rise to the signal at m/z 402.2 had a retention time similar to that of Gly-NC100194, and tandem mass spectrometry (MS/MS) spectra of this substance showed exactly the same pattern as obtained with Gly-NC100194 (Fig. 3, A and B). Thus, Gly-NC100194 was identified as a metabolite in this human urine sample similar to that reported for rats (Skotland et al., 2006). Furthermore, the MS/MS data in Fig. 3, A and B, indicated that the weak signal at m/z 303.3 in the postdose urine (Fig. 2B) was caused by a fragment of Gly-NC100194 formed in the MS. The fragmentation pattern for Gly-NC100194 is detailed in the rat metabolism report (Skotland et al., 2006).

View larger version (18K): [in this window] [in a new window]   FIG. 3. MS/MS spectra (mass range 150-500) of the main peaks observed in the inset chromatograms, i.e., following fragmentation in the MS of the ions giving rise to the signal at m/z = 402.3 ± 1.0 of the postdose urine sample shown in Fig. 2B (A) and synthesized standard Gly-NC100194 (B) and at m/z = 345.5 ± 1.0 of the same postdose urine sample (C) and synthesized standard NC100194 (D).

Gly-NC100194 and NC100194 were observed as metabolites in urine samples from all the subjects receiving the highest dose (Table 1). No other metabolite signal was identified in any of these subjects (data not shown), which was as expected based on the data obtained in the rat study.

Estimation of Gly-NC100194 and NC100194 in Urine Samples. The urine samples analyzed with their respective collection periods (up to approximately 13 h) and volumes for all the subjects of the highest-dose group are listed in Table 1. As shown in this table, the highest concentrations of both metabolites were observed in the urine samples collected during the first hour after injection. Moreover, in the samples collected during the first hour after injection, the amount of NC100194 was only 4 to 8% of that of Gly-NC100194 for six of the eight subjects. This ratio increased with time after injection for all eight subjects. Considerably higher ratios of NC100194 to Gly-NC100194 were obtained in the samples from subjects 42 and 45 than from the other six subjects. It should be noted that the extreme high ratios obtained for subject 45 were in part caused by interferences from an endogenous substance (also observed in the predose sample) resulting in an overestimation of the concentration of NC100194 in these samples. Because the concentration of the interfering substance may vary between different urine samples, there is no good way of correcting for this interference, and the data for subject 45 are therefore reported as they were obtained, i.e., without any correction.

The data in Table 1 were used to calculate the total amount of each metabolite excreted in the urine samples. The accumulated recovery of Gly-NC100194 during the first 13 h after injection was in the range of 43 to 63% of the injected dose for seven of the eight subjects. In the samples from subject 45, the recovery of Gly-NC100194 was only 19% of the injected dose. This subject, who obviously metabolized NC100668 very fast, was the same subject who showed the endogenous urine substance that resulted in an overestimation of the amount of NC100194. The estimated accumulated amount of Gly-NC100194 for all eight subjects corresponds to 52 ± 18% (mean ± S.D.) of the injected dose of this part of NC100668. As mentioned above, two of the subjects showed a much higher recovery of NC100194 than for the six other subjects. Thus, a recovery of 33 and 56% of NC100194 was estimated for subjects 42 and 45, respectively (although this number is certainly overestimated for subject 45), whereas the six others showed a recovery of 3 to 10%. The estimated accumulated amount of NC100194 corresponds to 15 ± 19% (mean ± S.D.) of the injected dose when all eight subjects are included and 10 ± 10% if the data for subject 45 are not included in the calculations.

It should be noted that quantification of these metabolites was not possible in the rat urine samples (Skotland et al., 2006), although these samples contained much higher concentrations of the metabolites than the human urine samples. The difficulty in quantifying these metabolites in rat urine is probably a result of the much higher (and varying) concentration of both high- and low-molecular-weight substances in rat urine (Alt et al., 1985) and illustrates the differences in rat and human kidneys in handling proteins and peptides.

The recoveries for the two metabolites are listed in Table 1 together with the recovery of ^99mTc in the same samples. As shown in Table 1, the estimated sums of the two metabolites were in the range 46.2 to 86.4% of the injected dose, whereas the accumulated recoveries of ^99mTc in the same samples were in the range 50.6 to 91.4% of the injected dose. By looking at the data in Table 1 in more detail, it is clear that the estimated recoveries of the metabolites fit quite well with the ^99mTc data for many of the samples, e.g., all the samples analyzed for subject 42. On the other hand, there are rather large differences for some samples, e.g., the samples obtained during the first hour after injection for subjects 44, 49, and 50. The Gly-NC100194 levels estimated in these three samples were higher than expected from the radioactivity measurements. In addition, when comparing with the radioactivity measurements, there is a clear trend toward underestimation of the metabolite levels at very low metabolite concentrations. Because the radioactivity measurements are expected to be very accurate, the most likely explanation for this is the technical difficulty encountered in the estimation of the two metabolites as described under Materials and Methods. In summary, these data indicate that most of the ^99mTc excreted in urine is bound to either Gly-NC100194 or NC100194. Furthermore, our conclusion is supported by the published results on the in vivo (rat) and in vitro metabolic profile of ^99mTc-NC100668 (Edwards et al., 2006). Here it is concluded that the metabolites of ^99mTc-NC100668 are ^99mTc-Gly-NC100194, ^99mTc-NC100194, and ^99mTcO₄^-.

All the data reported above for analyses of metabolites in the urine samples were obtained from the subjects receiving the highest dose. Some analyses were also performed on urine samples collected from subjects in the next highest-dose group. Although Gly-NC100194 could be detected also in these urine samples (data not shown), the concentration of the metabolite was not estimated because of the high degree of uncertainty in these data.

The present data indicate that the metabolism in humans is very similar to that observed in rats as the two same metabolites were observed in a similar ratio in urine of both species. There are many proteases in the body that theoretically should be able to cleave peptides at the C-terminal side of Lys (and Arg) residues and thus be able to cleave Gly-NC100194 from NC100668. As discussed in the rat metabolism study (Skotland et al., 2006), the "trypsin-like protease" present in large amounts in the brush-border membrane (Guder and Ross, 1984) is an obvious candidate for cleaving Gly-NC100194 from NC100668, and the NC100194 observed in urine is most likely formed from Gly-NC100194 by an attack of membrane alanyl aminopeptidase (EC 3.4.11.2 [EC] ), which is present in large amounts on the kidney brush-border membrane (Turner, 1998).

In conclusion, analysis of blood and urine samples obtained following injection of ^99mTc-NC100668 in healthy volunteers revealed a rapid elimination from blood and mainly urinary excretion fitting with a mechanism of glomerular filtration. The substance seems to be metabolized similarly in rats and humans because the same two C-terminal metabolites were detected in urine samples from both species.

	Footnotes

 
doi:10.1124/dmd.106.014126.

ABBREVIATIONS: LC/MS, liquid chromatography/mass spectrometry; SPE, solid-phase extraction; LOQ, limit of quantification; MS/MS, tandem mass spectrometry.

Address correspondence to: Kim Toft, Research and Development, GE Healthcare, Nycoveien 2, N-0401 Oslo, Norway. E-mail: kim.toft{at}ge.com

	References

Top Abstract Materials and Methods Results and Discussion References

 


Alt JM, Maess B, and Hackbarth H (1985) Species and strain differences in urinary protein excretion. Renal Physiol 8: 301-309.[Medline]

Edwards D, Battle M, Lear R, Farrar G, Barnett DJ, Godden V, Coombes C, Oliveira A, and Ahlström H (2006) The in vivo and in vitro metabolic profile of ^99mTc-NC100668, a new tracer for imaging venous thromboembolism: identification and biodistribution of the principal radiolabeled metabolite. Drug Metab Dispos 34: 1128-1135.[Abstract/Free Full Text]

Guder WG and Ross BD (1984) Enzyme distribution along the nephron. Kidney Int 26: 101-111.[Medline]

Liu S, Edwards DS, and Barrett J (1997) ^99mTc labelling of highly potent small peptides. Bioconjug Chem 8: 621-636.[CrossRef][Medline]

Skotland T, Hustvedt SO, Oulie I, Jacobsen PB, Friisk GA, Langøy AS, Uran S, Sandosham J, Cuthbertson A, and Toft KG (2006) NC100668, a new tracer for imaging of venous thromboembolism: disposition and metabolism in rats. Drug Metab Dispos 34: 111-120.[Abstract/Free Full Text]

Toft KG, Oulie I, and Skotland T (2005) Quantification of NC100668, a new tracer for imaging of venous thromboembolism, in human plasma using reversed-phase liquid chromatography with electrospray ion-trap mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 829: 91-96.[CrossRef][Medline]

Turner AJ (1998) Membrane alanyl aminopeptidase, in Handbook of Proteolytic Enzymes (Barret AJ, Rawlings ND, and Woessner JF eds) pp 996-1000, Academic Press, San Diego.

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.106.014126v1 35/11/1979    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Toft, K. G. Articles by Skotland, T. Search for Related Content PubMed PubMed Citation Articles by Toft, K. G. Articles by Skotland, T.