Neodymium-140 DOTA-LM3: Evaluation of an In Vivo Generator for PET with a Non-Internalizing Vector

140Nd (t1/2 = 3.4 days), owing to its short-lived positron emitting daughter 140Pr (t1/2 = 3.4 min), has promise as an in vivo generator for positron emission tomography (PET). However, the electron capture decay of 140Nd is chemically disruptive to macrocycle-based radiolabeling, meaning that an in vivo redistribution of the daughter 140Pr is expected before positron emission. The purpose of this study was to determine how the delayed positron from the de-labeled 140Pr affects preclinical imaging with 140Nd. To explore the effect, 140Nd was produced at CERN-ISOLDE, reacted with the somatostatin analogue, DOTA-LM3 (1,4,7,10- tetraazacyclododecane, 1,4,7- tri acetic acid, 10- acetamide N - p-Cl-Phecyclo(d-Cys-Tyr-d-4-amino-Phe(carbamoyl)-Lys-Thr-Cys)d-Tyr-NH2) and injected into H727 xenograft bearing mice. Comparative pre- and post-mortem PET imaging at 16 h postinjection was used to quantify the in vivo redistribution of 140Pr following 140Nd decay. The somatostatin receptor-positive pancreas exhibited the highest tissue accumulation of 140Nd-DOTA-LM3 (13% ID/g at 16 h) coupled with the largest observed redistribution rate, where 56 ± 7% (n = 4, mean ± SD) of the in situ produced 140Pr washed out of the pancreas before decay. Contrastingly, the liver, spleen, and lungs acted as strong sink organs for free 140Pr3+. Based upon these results, we conclude that 140Nd imaging with a non-internalizing vector convolutes the biodistribution of the tracer with the accumulation pattern of free 140Pr. This redistribution phenomenon may show promise as a probe of the cellular interaction with the vector, such as in determining tissue dependent internalization behavior.

electron capture with no emission of gamma photons (Figure 1) (6). Because 140 Pr has a 51% positron branch (Emean = 1.07 MeV) and a short half-life, the pair has potential for long-lived positron emission tomography (PET) tracing of pharmaceuticals. Together as a so-called in vivo generator (7,8), they provide a high positron yield with lanthanide labeling chemistry and a parent half-life that is suitable for monoclonal antibody, nanoparticle, and peptide imaging (9,10). In this light, it is interesting to pursue development of 140 Nd to investigate how the delayed positron emission from 140 Pr affects medical imaging, and how it can be exploited. The radionuclide 140 Nd is non-standard in radiopharmacy, but can be produced in a variety of methods: via (p,2n) reactions on naturally monoisotopic praseodymium-141 (11), 3 He bombardment of natural cerium (11)(12)(13), or by spallation processes on tantalum (14). Due to the relatively lower volatilization temperature of the rare earths (compared to tantalum), it is possible to extract the spallation-induced radiolanthanides by thermal diffusion and separate them by mass, e.g., at the on-line separator ISOLDE at CERN (15,16). In principle, this leads to the highest possible specific activity for radiochemistry and has been successfully employed in previous experiments with radiolanthanides (3,17,18). The (p,2n) production method is attractive for future developments because it is a reaction that is reachable by biomedical and hospital cyclotrons. However, it requires very high purity praseodymium starting material (without neodymium contamination), and a robust lanthanide separation technique in order to achieve radiolabeling with high specific activity.
When considering 140 Nd for PET, its value for direct imaging hinges upon the chemical and kinetic profile of the positron producing daughter nuclide 140 Pr. Previous reports show that the EC decay of DOTA-bound 140 Nd is highly efficient at releasing the daughter 140 Pr from the chelate, making it available for further interactions as a Pr 3+ cation (13). Praseodymium and neodymium have remarkably similar chemistry, and under the right conditions Pr 3+ could be predicted to re-bind a free chelator after being released. However, an activation barrier to stable binding between lanthanide ions and DOTA precludes room temperature chelation of the Pr 3+ daughter. Other chelators such as DTPA are not inhibited by an activation barrier and may fare better at retaining the daughter praseodymium. The mobility of the daughter praseodymium determines how different the distribution of the tracer-bound parent is from the distribution of the unbound daughter, the evaluation of which will determine the value of 140 Nd/ 140 Pr with functionalized DOTA in vivo. The current set of experiments serves as a preliminary investigation into the 140 Nd/ 140 Pr in vivo PET generator.
In order to create the appropriate scenario to test 140 Nd/ 140 Pr, the somatostatin receptor was selected as the target. The reason for this is threefold. First, somatostatin analogs are already employed clinically with the therapeutic radionuclide 177 Lu (19) and the diagnostic radionuclides 64 Cu (20) and 68 Ga, where a long-lived positron emitting lanthanide could prove useful in dose determinations. Second, the depth of research into somatostatin receptors has led to the development of well-established internalizing vectors (21), such as DOTATATE, and non-internalizing vectors (22) such as DOTA-LM3 (23,24). And third, the receptor is expressed in the pancreas, but not in many other tissues: thereby providing a test-tissue with more realistic perfusion than xenograft tumors. In the present study, testing was performed with DOTA-LM3 anticipating that the redistribution of praseodymium would be most evident with a targeting vector that remained located on the surface of the targeted cells.
Herein, we present the results from 140 Nd-DOTA-LM3 PET quantifications in H727 xenograft tumor-bearing mice before and after euthanasia. The pre-and post-mortem images represent the daughter and parent radionuclide distributions, respectively. As positrons are only emitted by the daughter, PET scanning only reveals the parent distribution in the absence of biological processes that differentiate the vector bound parent from the daughter. We also show verification of the dislocation (also referred to as de-labeling) of 140 Pr from DOTA-LM3 by radio-HPLC. Furthermore, ex vivo biodistributions from 140 Nd-DOTA-LM3 and 140 Nd as the free ion are used to show the source and sink organs for the free praseodymium daughter.

MaTerials anD MeThODs general
All water was 18 MΩ-cm MilliQ purified and was used to produce all aqueous solutions. Hydrochloric acid solutions were prepared from concentrated HCl (TraceSelect, Sigma).

Production of 140 nd
A 55 g/cm 2 tantalum foil target was irradiated by a 1.4 GeV proton beam, creating a multitude of radioactive and stable spallation products. The product nuclei, lanthanides in particular, diffused from the ≈2,000°C target to a ≈2,000°C tungsten surface ionizer. The ions were extracted at 30 kV and mass-separated with a 70° sector magnet (1.5 m mean bending radius). The A = 140 beam was implanted into two Zn-coated gold foils. The zinc, totaling 2-3 mg had been electrodeposited onto the gold foils over approximately 0.5 cm 2 . The entire procedure is nearly identical to the methodology recently described for projects collecting Tb isotopes at ISOLDE (3,17).

radiochemistry
The following methodology was carried out two times with small variations between each run. The zinc layer containing 140 Nd was etched briefly with aq. HCl (200 µL, 2 M), and the resulting solution was diluted to 2.2 mL with aq. HCl (2 M). This was heated to 98°C, cooled, and passed over AG1x8 anion exchange resin (300 mg, Biorad, 200-400 mesh, initially formate-form) packed in a 4 mm internal diameter (ID), fritted polypropylene column (Supelco) that had been prepped by three times sequential washing with water (3× bed volume), 2 M HCl (3× bed volume), and 6 M HCl (3× bed volume), finishing with equilibration in 2 M HCl. The resin has a high affinity for [ZnCl4] 2− ions in 2 M HCl (25) and was intended to remove any zinc impurity from the 140 Nd. An additional 0.5 mL of 2 M HCl was used to rinse residual 140 Nd from the column, and the entire effluent was collected and adjusted to pH 5-6 with aq. ammonium acetate (1 M, pH 7) and aq. ammonium hydroxide (28%, TraceSelect, Sigma) to a final acetate concentration of roughly 200 mM. This solution was passed over a hydroxamate functionalized Waters CM resin bed (26) (100 mg, 4 mm ID) to trap the 140 Nd and was washed with water (7 mL), then eluted with aq. HCl (600 µL, 0.1 M). The eluent was kept as the stock 140 Nd solution for further radiolabeling and formulation.
For the DOTA-LM3 preparation, 220 µL of the 140 Nd stock solution was added to aq. ammonium acetate (780 µL, 300 mM). Two productions were made, the first with 9 µg DOTA-LM3 (added from a stock solution of 1 mg/mL in water) and the second with 18 µg DOTA-LM3. The solution was heated in a sealed container to 95°C and incubated for 25 min. After cooling to room temperature, the labeling reaction was quenched with aq. DTPA (12 µL, 0.5 mM, pH 7) and let stand for 5 min. The solution was passed over a Waters C18 light sep-pak (prepped with 10 mL ethanol and 10 mL water) to trap the labeled product, rinsed with 1 mL water, and then eluted with 2 mL ethanol. The ethanol solution was taken almost to dryness (residual volume was approximately 50 µL) with the residual being diluted with 900 µL HEPES buffered isotonic saline (10 mM HEPES, 150 mM NaCl, pH 7.4) and was used directly for injections.
hPlc Verification of De-labeling and radioTlc for Determination of radiochemical Purity Samples of 140 Nd-DOTA-LM3 were injected onto a reverse-phase C-18 (Luna 3uC18(2)(n) 100 A 100 × 2 mm 3 μm, Phenomenex) column at a flow rate of 0.5 mL/min starting from 0% acetonitrile in water, and reaching 100% over a 15 min gradient. Elution was monitored with a radio detector. The entire effluent was collected in 1 min intervals (500 µL each) and quantified 4 days after collection by liquid scintillation counting on a HIDEX 300 SL spectrometer.

PeT imaging and Ex Vivo Biodistributions
NCI-H727 lung carcinoid cancer cells (ATCC CRL-5815, LGC Standards) were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Invitrogen) at 37° C and 5% CO2. Cells in their exponential growth phase and at 80-90% confluence were harvested by trypsinization and resuspended in 1:1 media and matrigel (BD Biosciences) at 5 × 10 7 cells/mL. Subcutaneous tumors were established in female NMRI nude mice (Taconic, Denmark) by inoculation of 5 × 10 6 cells in 100 µL on each flank above the hind limbs in the subcutaneous space. All animal experiments were performed under a protocol approved by the National Animal Experiments Inspectorate of Denmark.
Longitudinal small animal PET/CT imaging (Inveon Multimodality PET/CT scanner, Siemens) was performed with NCI-H727 tumor bearing mice injected intravenously with 3.3-4.3 MBq 140 Nd-DOTA-LM3 (n = 8) or 2.7-3.1 MBq 140 Ndchloride (n = 3) in 150 µL. Mice were anesthetized with sevoflurane (Abbott Laboratories) during injection and PET/CT imaging. PET data was acquired for 600 s in list mode at 1, 3, and 16 h after injection. The mice were sacrificed after the 16 h time-point and a PET acquisition was performed 2 h post-mortem. Images were reconstructed using a 3D maximum a posteriori algorithm with CT based attenuation correction. CT images were acquired with the following settings: 300 projections, 65 kV, 500 µA, and 400 ms exposure, and reconstructed with an isotropic voxel size of 105 µm. Image analysis was performed using the Inveon Software (Siemens). Region of interests (ROIs) were drawn manually over the tumor regions and other organs based on the CT images and the uptake of 140 Nd-DOTA-LM3 or 140 Nd-chloride quantified as % injected dose per gram tissue (%ID/g).
Conventional ex vivo biodistribution was performed after the post-mortem scan. Tumors and organs were resected, weighted and the radioactivity was counted in a gamma counter (Wizard 2 , PerkinElmer). resUlTs anD DiscUssiOn isolation of 140 nd at isOlDe, radiochemical Purification, and radiolabeling of 140 nd-DOTa-lM3  The eluted 140 Nd was reacted with DOTA-LM3 in ammonium acetate buffer (300 mM, pH 4.8), and after 30-60 min at 95°C radioTLC indicated that 140 Nd-DOTA-LM3 had formed in a 75% radiochemical yield. Quenching with DTPA and C-18 sep-pak purification led to an ultimate combined radiochemical yield and recovery of 60% in HEPES-buffered saline (pH 7.4). RadioTLC after C-18 purification indicated >95% radiochemical purity of 140 Nd-DOTA-LM3. The production, purification, and labeling procedure was performed twice, where the amount of peptide relative to radioactivity was selected based upon titration. Although it would have been ideal to have all samples at identical specific activity, it was not possible, and in this case, the final radiolabeled specific activities for 140 Nd-DOTA-LM3 were 5.0 and 2.5 MBq/nmol, each with sufficient activity for injection of four mice (see below). 140 Nd in an unchelated form was prepared for injection by pH adjustment of the eluted 140 Nd stock solution with sodium HEPES and diluted to a final formulation in pH 7.4, 150 mM NaCl, and 10 mM HEPES. Characterization of the 140 Nd chemical species was not performed, and it is herein referred to as " 140 Nd-chloride" for convenience.

hPlc-Traces of 140 nd DOTa-lM3
Purified 140 Nd-DOTA-LM3 was analyzed on reverse-phase HPLC to highlight the parent-daughter dechelation effect. The relative quantifications of the HPLC effluent are shown overlaid in Figure 2. The product 140 Nd-DOTA-LM3 eluted at 7.9 min, which agreed with the equilibrated liquid scintillation counter (LSC) trace, confirming >95% radiochemical purity of the 140 Nd-DOTA-LM3. It should be noted that parent-daughter transient equilibrium was reached before the LSC samples were counted, meaning that the LSC signal was representative of the parent 140 Nd elution profile. In contrast, the online radiodetector trace demonstrated the daughter 140 Pr behavior. This is because the detector was more sensitive to gamma radiation arising from positron annihilation after 140 Pr decay, and because of the relatively low abundance of penetrating radiation arising from 140 Nd decay. Here, 140 Pr was shown to elute with the solvent front with an elevated baseline between the solvent peak and elution of the parent 140 Nd-DOTA-LM3. Since the 140 Pr 3+ eluted faster than the radiolabeled peptide, the solvent front peak was evidence of the formation of 140 Pr 3+ in the injected solution, and the elevated baseline showed the in situ formation and washout of 140 Pr 3+ from 140 Nd decaying on the column. From the trace, it was evident that the release of 140 Pr from DOTA-LM3 after 140 Nd decay is >95% efficient, matching the observations of Zhernosekov and co-workers (13). Furthermore, the column behavior illustrated the expected in vivo behavior: a rapid redistribution of the positron-emitting daughter after the decay of the parent.
Ex Vivo Biodistributions of 140 nd-DOTa-lM3 and 140 nd-chloride Eight mice bearing dual-flank NCI-H727 lung carcinoid tumors were injected with 3-4 MBq 140 Nd-DOTA-LM3: four at a specific activity of 5 MBq/nmol and four at 2.5 MBq/nmol. A further three tumor-bearing mice were injected with 3 MBq 140 Nd-chloride. PET quantifications were obtained at 1, 3, and 16 h postinjection. After the last scan the animals were euthanized, and following equilibration of the daughter, the mice were rescanned. Finally, the animals were dissected, and tissue samples were weighed and counted.
For the unbound 140 Nd-chloride injections, the ex vivo biodistribution (16 h) showed high levels of accumulation in the lungs, spleen and liver, and to a lesser extent bone and tumor (Figure 3). This is a typical biodistribution for free +3 oxidation state radiolanthanides [for femur and liver vs. tumor, see, Ref. (27)] and for other hard radiometals [e.g., see, Ref. (24)]. The tissues with high accumulation of 140 Nd were also expected to accumulate released 140 Pr, owing to the chemical similarities between praseodymium and neodymium. Thus, this biodistribution serves as a descriptor of which tissues we expect to exhibit "sink" behavior in the pre-mortem PET scans.
The ex vivo biodistribution of 140 Nd-DOTA-LM3 was very distinct from that of 140 Nd-chloride with the pancreas, a known somatostatin receptor positive organ, being particularly interesting (21). The pancreas was found to take up 140 Nd-DOTA-LM3 without accumulating the free radiometal (13% ID/g with high specific activity 140 Nd-DOTA-LM3, compared to 0.1% ID/g with the free 140 Nd 3+ ). Therefore, in the PET study, the pancreas was expected to have a lower signal in the pre-mortem 140 Nd-DOTA-LM3 scans compared to the post-mortem scans. The liver, spleen, and lungs had the opposite behavior, accumulating the free radiometal but not the peptide, and were expected to have a higher signal in the pre-mortem scans than in the post-mortem.
Fani et al. showed that in comparing the biodistribution of 68 Ga-NODAGA-LM3, 68 Ga-DOTA-LM3, 64 Cu-NODAGA-LM3, and 64 Cu-CBTE2A-LM3, tumor uptake relative to the pancreas, stomach, and kidney is highly variable depending on the metal and chelator used (23,28). In the current work, we injected 300 pmol (5 MBq/nmol) or 600 pmol (2.5 MBq/nmol) in order to get enough signal for the PET scans. For the biodistributions from Fani et al., 10 pmol was injected, and blocking was performed with 200 nmol of excess DOTA-LM3. The overall effect of the blocking was to reduce the uptake in sst2 expressing tissues, which is exactly what was observed between the higher and lower specific activity injections of the current study: a modest reduction in the uptake in sst2 expressing tissues relative to the kidney, and overall faster excretion.
Based upon the ex vivo biodistribution, the tumors had an intermediate behavior, weakly concentrating both the free 140 Nd and the labeled peptide, meaning that the PET signal was expected to change little between the pre-and post-mortem scans (i.e., washout of 140 Pr from the tumor volume could be compensated by uptake of 140 Pr from the bloodstream). This behavior is in many ways an undesirable outcome, because signals arising from the tumors can be attributable to both the free metal ions and the targeted peptide. Future work with a different vector/ target system could give a result with a simpler interpretation. Nevertheless, the pancreas remains an interesting tissue within the present set of experiments.
PeT studies show Tissue Dependent redistribution of 140 Pr after 140 nd Decay Positron emission tomography data were analyzed to quantify the 140 Pr signal from the tumors, pancreas, kidney, lung, and liver ( Table 1). The PET signals for the tumors are given as the percent of the injected signal per gram (%IS/g) (Figure 4). This unit is non-standard and is described further in the Supplementary Material. %IS/g was chosen for two reasons: first because the PET scans quantify the redistributed daughter 140 Pr, not the 140 Nd-tracer, and second because the data were not corrected for point spreading due to extensive positron range. This means that there is a substantial partial volume distortion when converting from annihilation signal (%IS/g) to tracer concentration (%ID/g), and that the well-counter based ex vivo biodistribution is not directly comparable to the post mortem PET results. A discussion of the partial volume effect due to the high energy positrons from 140 Pr is included in the Supplementary Material. The tumor time-activity curve shows that in this model (as expected from the ex vivo biodistribution) very little redistribution is observed. For the present case, the tumor was known to accumulate the trivalent lanthanide to some small degree (from the Nd-chloride injection data above), meaning that 140 Pr 3+ produced by decay in the tumor region had some tendency to remain. In other organs, however, due to the dechelation effect, there was a dramatic change in the PET signal in the pre-and post-mortem imaging quantifications. The redistribution effects are displayed in Figure 5 as the ratio of the mean organ signal between the post-mortem, and pre-mortem (16 h) images. This effect is most evident in the pancreas and liver for the high specific activity injections, where the decrease in liver signal is matched by an increase in the pancreas signal, confirming the behaviors of the peptide and free metal observed in the ex vivo biodistribution.
It should be noted that the injected mass of the tracer had a large effect upon the PET quantifications and ex vivo biodistribution (Figures 4 and 5). This indicates that the receptorspecific accumulation was becoming saturated as the injected mass increased from 0.5 nmol (5.0 MBq/nmol) to 1 nmol (2.5 MBq/nmol). This saturation behavior is non-desirable for probing the internalization behavior of the peptide as nonspecific interactions begin to dominate the tracer distribution. Figure 6 shows an example PET/CT reconstruction, qualitatively illustrating the redistribution effect. The most striking differences in the images between the daughter distribution (16 h, left panels) and the parent distribution (post-mortem, right panels) are seen in the liver, lungs, and pancreas. As expected, the 16 h pre-mortem PET images more closely reflected the biodistribution observed for the free 140 Nd-chloride injections, while the post-mortem images showed the biodistribution for the intact 140 Nd-DOTA-LM3 tracer.
The most important result from the PET imaging was the pancreatic signal. In this case, in the pre-mortem image, it was

3.47
The tissue signal quantifications in %IS/g are given for the 16 h postinjection scans (16 h)   studies show that 56 ± 7% (n = 4, mean ± SD) of the in situ produced 140 Pr washed out of the pancreas before decay. For statistical analysis the pre-and post-mortem PET quantifications were compared with a paired-difference two-tailed t-test. The tabulated data are presented in Table 1, along with the p values. In this case, the t-test was used to determine the significance of the absolute difference between the pre-and post-mortem signals in %IS/g. Admittedly, there are many ways to analyze these data, and in this case, the paired-difference test was selected because it adds to the statistical power by comparing the tissues in a single subject to themselves after intervention. Clearly from the p values in Table 1, the use of DOTA-LM3 as a tracer led to significant changes from the pre-to post-mortem images, while the non-targeted 140 Nd-chloride remained largely unaltered. Of note in the free ion 140 Nd-chloride injections, is that the only tissue with a statistically significant difference between the pre-and post-mortem quantification is the kidney, whereas in the targeted DOTA-LM3 images the only tissue lacking a significant difference was the muscle. The kidney is interesting because in all cases the post-mortem signal was higher, by 25-50% than the pre-mortem value. While it could be suggested that this is due to rapid excretion of 140 Pr 3+ from the kidney to the bladder in vivo, the fact that this is also observed with the 140 Ndchloride injections indicates that within the kidney the chemical form of the neodymium is not necessarily as a free cationic lanthanide. However, in all other tissues, the distribution of 140 Nd from the 140 Nd-chloride injections strongly resembles that of redistributed 140 Pr.
The pancreatic washout reveals a potential benefit derived from the 140 Pr's delayed positron. Specifically, the degree of redistribution of 140 Pr 3+ may be affected by its location and access to blood flow. This means that the PET signal observed with 140 Nd labeled vectors might be highly dependent on their cellular internalization status, as 140 Pr 3+ cations originating from decays occurring on the surface of the cell or in circulation may be transported away by the blood flow, whereas 140 Pr 3+ cations released from 140 Nd decay inside of a cell have an additional diffusion barrier. A conceptual graphic demonstrating the idea is depicted in Figure 7. As many promising new classes of pharmaceuticals, in particular nanoparticle drug formulations, gene therapy and targeted Auger emitting radionuclides, are expected to be most effective when internalized, a PET radiolabel for determining internalization would be a valuable tool for drug development.

general Discussion
When searching for long-lived PET radionuclides, the utility of 140 Nd is immediately evident in its half-life and lack of concurrent gamma emissions. However, due to the nature of the delayed positron from the short-lived daughter, it is important to understand how imaging may be affected by dechelation. The redistribution of 140 Pr in the present case was clearly visible in the pre-and postmortem PET images. While the tumor signal was significantly changed, the magnitude of change was small which may preclude application. However, this behavior might be model dependent, and not general for all tumor types or locations. Nevertheless, the signals from the other tissues show the potential for using the daughter-delay to determine the in vivo internalization status of new probes, as highlighted by the pancreatic signal. These data support a hypothesis that in certain cases, PET imaging with 140 Nd provides a localized signal only if a vector is internalized. This capability may prove useful in future drug development where in vivo internalization is critical for drug action. The resulting atomic rearrangement releases the daughter 140 Pr from DOTA as a free ion. If 140 Pr diffuses into the blood stream before decaying (upper path), it will be carried away and give a remote positron emission tomography (PET) signal. However, if there is a diffusion barrier such as a cell membrane or a lack of blood flow (lower path), the PET signal will remain localized to the tracer accumulation site. Overall, the statistical analysis proves that the images generated using a DOTA-based 140 Nd/ 140 Pr in vivo generator are significantly altered from the true distribution of the tracer. With more development, it may be possible to use this technique to determine specific details of the interaction between the tracer and its molecular target. While the p-values are often less than the nominal 0.05 designation for statistical significance, practical significance is weakened by a large inter-subject variability. This means that further development in the 140 Nd in vivo generator system will, for the time-being, remain an invasive procedure relegated to pre-clinical drug development.

cOnclUsiOn
In this study, we showed that the non-internalizing tracer 140 Nd-DOTA-LM3 accumulates in the pancreas and releases 140 Pr 3+ into the blood stream where it quickly redistributes to the liver and lungs. We hope that further work will lead to the development of internalization sensitive PET probes using 140 Nd as the radiolabel. The experimental set up described here with pre-and post-mortem imaging should facilitate that development as it allows direct quantification of the parent ( 140 Nd, post-mortem) and daughter ( 140 Pr, pre-mortem) in the same subject. The ability to determine the tissue-dependent internalization of pharmaceuticals using PET would aid greatly in drug delivery designs where cellular internalization is crucial to drug action.