Abstract
The application of 123I-3-iodo-α-methyltyrosine is limited to diagnosis of brain tumors due to its marked long-term uptake in kidneys. In vitro evaluation of 125I-2-iodo-l-phenylalanine showed high uptake in R1M cells by L-type amino acid transport system 1 (LAT1). This study evaluates 123I-2-iodo-l-phenylalanine as a new specific tumor tracer for SPECT. Methods: 123/125I-2-Iodo-l-phenylalanine is prepared as a one-pot kit using the Cu1+-assisted isotopic exchange method. The characteristics of 125I-2-iodo-l-phenylalanine were examined in vivo in R1M tumor–bearing athymic mice and in acute inflammation–bearing NMRI mice. The uptake of 123/125I-2-iodo-l-phenylalanine in tumor and other organs of interest was measured by dynamic planar imaging (DPI) and γ-counting after dissection. Displacement of 123I-2-iodo-l-phenylalanine radioactivity by l-phenylalanine, l-methionine, and l-cysteine was measured. 123I-Iodo-human serum albumin planar imaging was performed to correct for blood-pool activity and MRI was performed to delineate the tumor in DPI. 18F-FDG uptake was measured with an animal PET scanner. 125I-2-Iodo-l-phenylalanine and 18F-FDG uptake in inflamed muscle were compared. Results: 123/125I-2-Iodo-l-phenylalanine showed a high and fast tumor uptake and followed a reversible first-order pattern allowing calculation of the half-life and the time to reach equilibrium (tR). Net tumor-to-background ratios up to 6.7 at 60 min were obtained. This radioactivity was significantly displaced by l-phenylalanine, l-methionine, and l-cysteine, pointing to reversible LAT transport. When plotting tR of the tumor uptake as a function of tumor volume, a rectangular hyperbolic curve was obtained. The almost constant tR values at higher tumor volumes (>4 mL) could be linked to increased necrotic tissue. Fast blood clearance of the tracer through the kidneys to the bladder and low tracer activity in the abdomen and brain were observed. The inflamed muscle showed only a slight increase of 125I-2-iodo-l-phenylalanine uptake (inflammation-to-background ratio, RIB = 1.30 ± 0.02), in contrast to the high 18F-FDG uptake (RIB = 11.1 ± 1.7). The in vivo stability of 123/125I-2-iodo-l-phenylalanine was good: Only 7% of free radioiodide and no other labeled metabolites were observed after 90 min. Conclusion: 123/125I-2-Iodo-l-phenylalanine is quickly taken up by the overexpressed LAT1 system in R1M tumors with high tumor specificity. The availability of a kit and the specificity of the tracer make 123I-2-iodo-l-phenylalanine a promising tool for oncologic SPECT.
Both amino acid transport across the cell membrane and the protein synthesis rate are early features of malignant transformation. A- and L-type amino acid transport has been shown to be increased in tumor cells relative to normal tissue, and these transport systems have been the major focus of the development of amino acid tumor tracers for oncologic imaging (1,2).
At present, 123I-3-iodo-α-methyltyrosine is the only routinely used amino acid tracer for SPECT tumor diagnosis. In comparison with 18F-FDG, this tracer has major advantages, such as high and fast tumor uptake and rather low uptake in gray matter and inflammatory lesions. However, its marked long-term renal accumulation limits its use for oncologic imaging outside the brain (2–5). Recently, other L-system–transported radiolabeled amino acids for SPECT have been developed and evaluated to overcome this problem: 2-iodo-l-tyrosine (6), 4-iodo-l-phenylalanine (7), and 2-iodo-l-phenylalanine. The latter was developed in our research group and showed high accumulation in vitro by L-type amino acid transport (more specifically, LAT1) in R1M (rhabdomyosarcoma) tumor cells without incorporation in the cell proteins (8). Recent publications verified that incorporation of radiolabeled amino acids is not necessary and that the increased uptake is directly related to the metabolic requirements of the tumor cells (8). Therefore, we evaluated in vivo, in the rodent, 123I-2-iodo-l-phenylalanine as a potential SPECT tracer to detect brain as well as peripheral tumors.
MATERIALS AND METHODS
All of the conventional products used were at least analytic or clinical grade and were obtained from Sigma-Aldrich. The solvents were of high-performance liquid chromatography quality (Chemlab).
Synthesis of Precursor 2-I-l-Phenylalanine
2-I-l-Phenylalanine was prepared from 2-Br-l-phenylalanine (PepTech Corp.) using Cu1+-assisted nucleophilic exchange under acidic and reducing conditions (30.3 mmol/L 2-Br-l-phenylalanine, 4.4 mmol/L CuSO4, 8.9 mmol/L citric acid, 9.0 mmol/L SnSO4, 10.7 mmol/L gentisic acid, 44.5 mmol/L NaI) at 160°C for 16 h (9). 2-I-l-Phenylalanine and 2-Br-l-phenylalanine were recovered separately from reversed-phase HPLC (RP-HPLC) separation (Hibar Lichrosorb RP-select 7 μm; λ = 261 nm; Merck) with 20:80 MeOH/H2O containing 1 mmol/L NH4Ac (pH 5.5, 13 mL/min), and 2-I-l-phenylalanine was purified from methanol by evaporation. Identification and quality control were achieved by NMR, liquid chromatography mass spectroscopy, thin-layer chromatography, and HPLC. Chiral chromatography (5-μm Chirobiotic T, 150 × 4.6 mm; Astec) with 20:80 MeOH/H2O containing 20 mmol/L NH4Ac (pH 5.5, 1 mL/min) was used to check chiral modification to 2-I-d-phenylalanine.
Radiochemistry
123I/125I-2-Iodo-l-Phenylalanine.
Radioiodination with 123I (222 MBq; 10–20 μL) or 125I (37 MBq; 10 μL) (Nordion Europe) of 1.0 mg 2-I-l-phenylalanine was performed by Cu1+-assisted isotopic exchange (9) under acidic and reducing conditions (0.2 mg CuSO4, 2.5 mg citric acid, 0.5 mg SnSO4, 1.3 mg gentisic acid in 565 μL; 60 min at 100°C). The reaction mixture was drawn up in a syringe containing the appropriate amount of 71 mmol/L trisodium citrate dihydrate to render the solution isotonic and to adjust the pH to at least 4. The reaction mixture was filtered through a 0.22-μm Ag filter (Millipore) and a sterile 0.22-μm filter (Millipore) into a sterile vacuum penicillin vial to remove the free 123/125I−. Identification and quality control were achieved by HPLC and Sep-Pak C18 (Waters). Chiral chromatography was used to check the absence of 2-I-d-phenylalanine.
123I-Iodo-Human Serum Albumin (HSA).
Radioiodination of HSA with 123I (Nordion Europe) was performed by electrophilic substitution using the IODO-GEN technique. IODO-GEN (1,3,4,6-tetrachloro-3α,6α-diphenylglycouracil; Pierce) was coated on the wall of polypropylene vials using 70 μg of IODO-GEN per 200 μL of chloroform per vial. The solvent was evaporated by air flow at room temperature. In the IODO-GEN vial, the 123I (111 MBq) solution was added to HSA (50 μg) in 140 μL 0.1 mol/L potassium phosphate buffer at pH 8.5. After 15 min at room temperature, the mixture was removed from the IODO-GEN–coated vial to stop the reaction and was filtered through a 0.22-μm Ag filter to remove the free 123I− from 123I-Iodo-HSA. The specific activity was 1.5 × 105 GBq/mmol. Quality control was achieved using a standard prepacked PD-10 SEC column (size-exclusion chromatography; Amersham Pharmacia Biotech) using phosphate-buffered saline/HSA as the eluting agent.
In Vivo Experiments
Laboratory Animals.
Water and food were freely available during the experimental period. For the tumor model, male SWISS∼nu/nu mice (n = 33) (Bioservices) were injected subcutaneously in the right flank (armpit region) with 5 × 106 R1M rhabdomyosarcoma cells. Tumors were grown and normal tumor growth curves were obtained using sliding caliper measurement and the estimate volume formula V = 0.4 a2·b, with a and b being the short axis and the long axis of the tumor, respectively (10). Imaging experiments with 123I-2-iodo-l-phenylalanine, 123I-HSA, and 18F-FDG and dissection experiments with 125I-2-iodo-l-phenylalanine were performed.
For the inflammation model, male NMRI mice (n = 9) (in-house breeding program) were injected in the right biceps brachii with 25 μL of turpentine. Imaging (123I-2-iodo-l-phenylalanine, 123I-HSA, and 18F-FDG) and dissection (125I-2-iodo-l-phenylalanine and 18F-FDG) experiments with the inflamed mice were performed 24 h after turpentine injection.
During all imaging experiments, the animals were anesthetized intraperitoneally with 75 μL (1.5 mg) of solution containing 20 mg pentobarbital per milliliter (Nembutal, 60 mg/mL; Ceva Santé Animale). For the biodistribution experiments by dissection, the animals were killed without sedation by cervical dislocation and the organs of interest were dissected.
All tracers (123/125I-2-iodo-l-phenylalanine, 18F-FDG, and 123I-HSA) were injected intravenously in the lateral tail vein. The study protocol was approved by the ethical committee for animal studies of our institution.
Dynamic Planar Imaging (DPI).
Imaging was performed using a γ-camera (Toshiba GCA-9300A/hg) in planar mode equipped with a high-resolution, parallel-hole collimator. All images were acquired into 128 × 128 matrices (field of view, 23.5 × 12.46 cm) and with a photopeak window set at 15% around 159 keV. Together with the animals, a syringe with a known amount of radioactivity was scanned, to allow the semiquantification of the results of the region-of-interest (ROI) analysis. The ROIs were drawn using an MRI maximum intensity projection (10). The ROI of the heart was used as a measure for blood-pool activity. Due to partial-volume effects, the measured blood-pool data will be overestimated (11). The injected dose (ID) was calculated as the amount of radioactivity in the syringe before and after injection (Capintec CRC-15R). The tracer uptake was expressed as the percentage ID per pixel (%ID/P).
At first, an 123I-HSA study was performed to measure the relative blood-pool distribution to correct the uptake of 123I-2-iodo-l-phenylalanine in the tumor and the inflammation area as well as the rest of the animal for blood-pool activity. Ten tumor-bearing mice and 3 acute inflammation–bearing mice were injected with 7.4 MBq 123I-HSA. Ten dynamic planar images of 1 min were acquired 10 min after injection. ROIs were drawn around the tumor or inflammation area and the contralateral background area. The tumor-to-contralateral background (RTB) and the inflammation-to-contralateral background (RIB) ratios were calculated and the mean for all animals was determined.
The DPI experiments with 123I-2-iodo-l-phenylalanine (Fig. 1) were started when the tumor reached a volume of 0.30 cm3. Thirty tumor-bearing mice were followed-up with the new tracer until the tumor reached a volume of 5.00 cm3.
Scheme of planar imaging procedure. (a) Static acquisition to determine background radiation. (b) DPI to determine plateau phase (equilibrium) for 123I-2-iodo-l-phenylalanine uptake. (c) DPI to determine displacement of 123I-2-iodo-l-phenylalanine radioactivity with l-phenylalanine, l-methionine, or l-cysteine. (d) Injection of 123I-2-iodo-l-phenylalanine at 0 min. (e) Injection of l-phenylalanine, l-methionine, or l-cysteine at 10 min.
DPI was started immediately after injection of 18.5 MBq 123I-2-iodo-l-phenylalanine. First, the steady state of tracer uptake in the tumor was determined. Second, at steady state, a displacement study with l-phenylalanine, l-methionine, and l-cysteine—which all use the L-type transport system (12)—was performed. For the displacement study, 200 μL of a 145 mmol/L amino acid solution was intravenously injected in the lateral tail vein.
ROIs were drawn around the tumor, the contralateral background area, the right kidney, the heart, the pancreas, the brain, the thyroid, and the total body. In the case of the inflammation model, the ROI for the tumor was replaced by the ROI for the inflammation site. Due to superposition of the pancreas with other organs such as left kidney and stomach, the results will be overestimated, especially during the beginning of the DPI scan.
Tumor 123I-2-iodo-l-phenylalanine uptake was compared with the uptake in the contralateral background area and the RTB was calculated. The significance of displacement of 123I-2-iodo-l-phenylalanine activity by the 3 amino acids was calculated for a 95% confidence interval.
The 123I-2-iodo-l-phenylalanine tumor uptake as a function of time, obtained from DPI experiments, were fit to a pseudo first-order model. The kinetic parameters half-life (t1/2), time to reach equilibrium (tR), and theoretic equilibrium for tumor tracer uptake were determined. The latter as well as the pseudo first-order fitting were performed in parallel with the in vitro experiments performed by Mertens et al. (8). The relationship of tumor volume as a function of tR was examined, nonlinear regression was performed, and a theoretic rectangular hyperbole was calculated.
In the case of the inflammation model, the uptake of 123I-2-iodo-l-phenylalanine in the inflammation area was compared with the contralateral background area and the RIB was calculated.
18F-FDG Animal PET.
PET was performed with a high-sensitivity animal PET scanner (VUB-PET; Vrije Universiteit Brussel; resolution, 3–4 mm) (13). Three NMRI mice bearing an acute inflammation and 3 R1M tumor–bearing athymic mice were injected with 18.5 MBq of 18F-FDG. Fifteen minutes after injection, one time frame of 15 min was acquired. The images were reconstructed using Fourier rebinning and 2-dimensional order-subsets expectation maximization (13). The images produced had a matrix size of 128 × 128 × 19.
Volumes of interest (VOIs) were drawn around the tumor (or inflammation site) using an MR image. The VOI for the tumor was horizontally mirrored to create the contralateral background VOI. The values are expressed as counts per voxel and the RTB and RIB were calculated.
MRI.
The tumor-bearing animals were anesthetized, and, after sedation, the animals were placed in a specially designed restrainer box in which sterile conditions could be retained. 3-Dimensional Dess-weighted images—hybrid T1–T2-weighted images—were generated on a 1-T whole-body system (Magnetom SP; Siemens) (10). The animals were scanned 2 at a time using a small surface flex coil.
Dissection: Biodistribution of 123I-2-Iodo-l-Phenylalanine.
The ID was calculated by weighing the syringes before and after injection of the tracer and by using a dilution series of the injected tracer solution that was also weighed and counted for radioactivity using the auto γ-counting system (CobraII Series; Canberra Packard).
Thirty R1M-bearing athymic mice were injected with 7.4 kBq 125I-2-iodo-l-phenylalanine, 6 d after the last imaging experiment was performed. At various time points (2, 30, 45, 60, and 180 min) after injection, 6 animals per time point were sacrificed. The organs and tissues were removed, washed, and weighted. The blood was collected in an ethylenediaminetetraacetic acid (EDTA)-coated vial. The radioactivity of the samples was counted by use of an auto γ-counting system (CobraII Series). The amount of radioactivity in the organs and tissues was calculated as the differential absorption rate (DAR): (%ID/g)tumor/(%ID/g)total body.
Inflammation-bearing NMRI mice (n = 3) were injected with 7.4 kBq of 125I-2-iodo-l-phenylalanine together with 16 MBq of 18F-FDG through the lateral tail vein. After 30 min, the mice were sacrificed and organs and tissues were removed, washed, and weighted. Muscle tissue was collected from the inflamed left leg and the noninflamed right leg. The 18F activity was counted on day 1 and the 125I activity in the same samples was counted 3 d later. The amount of radioactivity in the organs and tissues was calculated as the DAR.
Metabolism Study: In Vivo Stability of 125I-2-Iodo-l-Phenylalanine.
The blood, collected in the tumor model biodistribution study, was used for a metabolism study of 125I-2-iodo-l-phenylalanine as a function of time. Five hundred microliters of blood were centrifuged at 1,800g for 10 min, and the supernatant (plasma) was removed from the pellet. First, 200 μL of plasma were diluted 3 times with 9% NaCl. Second, 200 μL of this “plasma–NaCl solution” were added to the same volume of 20% trichloroacetic acid to denaturize the protein content. After a 5-min centrifugation at 1,800g, the supernatant was collected and filtered through a 0.22-μm filter. The samples were analyzed on RP-HPLC (C18 Hypersil BDS 5 μm; 250 × 46 mm; λ = 254 nm) (Alltech) with 20:80 MeOH/H2O containing 1 mmol/L NH4Ac (pH 5.5, 1 mL/min), and fractions of 0.5 mL were collected and counted by use of a γ-counting system (CobraII Series).
To determine the influence of EDTA on the formation of 125I-2-iodo-l-phenylalanine metabolites, 2 control experiments were performed. First, 7.4 kBq of 125I-2-iodo-l-phenylalanine were added to an EDTA-coated vial and treated identically to the blood samples. Second, mouse blood was recovered from a wild-type NMRI mouse and 7.4 kBq of 125I-2-iodo-l-phenylalanine were added. After 90 min of incubation at 37°C, the sample was treated as described earlier.
RESULTS
Synthesis and Radiolabeling of 2-I-l-Phenylalanine
Yields up to 65% were obtained for the presursor synthesis of 2-I-l-phenylalanine from 2-Br-l-phenylalanine using the Cu1+-assisted nucleophilic exchange method.
Radioiodination of 2-I-l-phenylalanine by Cu1+-assisted isotopic exchange, followed by Ag membrane filtration, resulted in a radiochemical purity of >99% and a specific activity of 65 GBq/mmol (123I labeling) and 11 GBq/mmol (125I labeling). In both cases, it was shown by chiral HPLC that there was no detectable amount of d analogs.
In Vivo Results
In Vivo Stability of 125I-2-Iodo-l-Phenylalanine.
HPLC analysis of the mouse blood showed a dehalogenation of 7.3% of 125I-2-iodo-l-phenylalanine 90 min after injection. No other metabolites were detected. EDTA did not show any influence on the deiodination of 125I-2-iodo-l-phenylalanine.
Planar Imaging.
DPI with 123I-HSA showed no significant difference between the blood flow in the tumor or inflammated muscle and the contralateral reference leg (RTB = 1.1 ± 0.2 and RIB = 1.1 ± 0.2).
The 123I-2-iodo-l-phenylalanine uptake–time kinetics in R1M tumors in vivo is shown in Figure 2 and follows a rectangular hyperbolic shape. When plotting 1/(net tumor uptake) as a function of 1/time, a linear relationship (R2 > 0.95) is obtained (Fig. 3A). This means that important kinetic parameters such as t1/2, velocity at initial conditions, and the theoretic equilibrium of tumor tracer uptake can be determined. This results in the calculated values for 123I-2-iodo-l-phenylalanine of (net tumor uptake)equilibrium = 2.7 %ID/P and t1/2 = 0.5 min. Figure 3B indicates that 123I-2-iodo-l-phenylalanine uptake in R1M tumors follows a pseudo reversible first-order reaction (R2 > 0.90) (8).
Overall mean uptake of 123I-2-iodo-l-phenylalanine uptake (%ID/P) in tumor and contralateral background region as functions of time by DPI for tumor volumes ranging from 0.3 to 5.0 cm3.
(A) Uptake of 123I-2-iodo-l-phenylalanine: plot of 1/(AAt) as function of 1/time. Equation of fitted curve: y = 0.17x + 0.37 (R2 = 0.9784) resulting in AAeq* = 2.73 %ID/P and t1/2 = 0.46 min. (B) Uptake of 123I-2-iodo-l-phenylalanine as function of time by DPI: pseudo first-order fit (R2 = 0.9764). *Net 123I-2-iodo-l-phenylalanine tumor uptake at equilibrium (%ID/P); ‡Net 123I-2-iodo-l-phenylalanine tumor uptake when equilibrium is not reached (%ID/P).
At equilibrium (plateau phase), net 123I-2-iodo-l-phenylalanine uptake in the tumor was high. It increased from 0.60 ± 0.03 %ID/P up to 5.7 ± 0.4 %ID/P for tumor volumes ranging from 0.3 to 5.0 cm3 (Figs. 2 and 4A and 4B). When plotting the 123I-2-iodo-l-phenylalanine tumor uptake at equilibrium as a function of tumor volume, we observed a large spread of the results (R2 = 0.11) and no correlation between tumor uptake and tumor volume. When plotting the time to reach equilibrium, tR, as a function of tumor volume, a hyperbolic curve (R2 > 0.90) reaching a plateau up from a tumor volume of 4.0 cm3 was obtained (Fig. 5). Nonlinear regression of a theoretic hyperbolic curve to the experimental values shows a top value of 15.3 ± 0.7 min. The experimental curve fitted the theoretic rectangular hyperbole (R2 = 0.91) (Fig. 5).
(A) Uptake of 123I-2-iodo-l-phenylalanine uptake in the rhabdomyosarcoma tumor model as function of time by DPI for tumor volume of 0.50 cm3. Each image is result of summation of 11 consecutive frames. Relative scaling was used (see colored tool bars) and each subpanel depicts 3 different animals for the time frame depicted at top of each subpanel. (B) Uptake of 123I-2-iodo-l-phenylalanine at steady state. Panel depicts last time frame (subpanel) shown in A and shows 3 different animals for time frame of 21–33 min after tracer injection. B = bladder; P = pancreas zone; K = kidney; T = tumor; C = calibration syringe.
Time to reach equilibrium (tR) as function of tumor volume. NLR = nonlinear regression.
The displacement of 123I-2-iodo-l-phenylalanine activity in the tumor with l-phenylalanine, l-methionine, and l-cysteine is summarized in Table 1. The radioactivity in the tumor decreased rapidly for a small but significant amount (P < 0.05) after intravenous administration of those amino acids and reached a new plateau within 2 min.
Displacement Study of 123I-2-Iodo-l-Phenylalanine Activity in Tumor Using Planar γ-Scintigraphy
123I-2-Iodo-l-phenylalanine was cleared quickly through the kidneys, without accumulation, to the bladder (Fig. 6). This is confirmed by the fact that the first-order fits of the activity as a function of time were almost the same for kidneys and the heart (Fig. 6). High tracer uptake (3.3 ± 1.5 %ID/P) was noted in the zone of the pancreas but, due to superposition of the pancreas with other organs such as the left kidney and the stomach, the results are overestimated (14). Low uptake of radioactivity in the thyroid (2.1 ± 1.1 %ID/P), brain (1.5 ± 0.8 %ID/P), or abdominal organs was observed (Fig. 7).
Uptake of 123I-2-iodo-l-phenylalanine uptake (%ID/P) as function of time by DPI: clearance of tracer through kidneys to bladder.
Uptake of 123I-2-iodo-l-phenylalanine uptake (%ID/P) in heart, pancreas, brain, and thyroid as function of time by DPI.
There was no significant (detectable) increased uptake of 123I-2-iodo-l-phenylalanine in the inflammation area. The RIB was 0.97 ± 0.14. This results in an apparent tumor–to–inflamed tissue ratio (T/I ratio) of 5.9 ± 0.42.
18F-FDG Animal PET.
The uptake of 18F-FDG was high in the tumor and also in the inflammation area. A RTB of 3.6 ± 1.6 and a RIB of 2.3 ± 0.6 were obtained. The resulting apparent T/I ratio is 1.6 ± 1.2.
Biodistribution by Dissection.
The biodistribution data of 125I-2-iodo-l-phenylalanine in R1M tumor–bearing athymic mice are shown in Table 2.
Biodistribution Study by Dissection of 123I-2-Iodo-l-Phenylalanine in R1M-Bearing Athymic Mice (n = 6)
The net 125I-2-iodo-l-phenylalanine tumor uptake reached equilibrium up from 60 min with a mean DAR of 3.8 ± 1.5. At the same time point, 125I-2-iodo-l-phenylalanine activity in the blood and in the contralateral leg tissue reached a DAR of 1.1 ± 0.3 and 0.6 ± 0.2, respectively.
125I-2-Iodo-l-phenylalanine was cleared very fast through the kidneys, without accumulation, to the bladder. A significant uptake of radioactivity in the pancreas was observed. No significant accumulation of radioactivity was observed in other abdominal organs, such as liver, lungs, small intestine, and large intestine, or in the brain.
The biodistribution data of 125I-2-iodo-l-phenylalanine and 18F-FDG in inflammation-bearing NMRI mice are shown in Table 3. A marked high 18F-FDG uptake in the inflamed muscle with a RIB value of 11.1 ± 1.7 is observed, whereas only a small increase of 125I-2-iodo-l-phenylalanine uptake in the inflammation site vis à vis the contralateral muscle was noticed with a RIB value of 1.30 ± 0.02. This results in a ratio T/I of 8.5 ± 1.7.
Biodistribution Study by Dissection of 125I-2-Iodo-l-Phenylalanine and 18F-FDG in NMRI Mice Bearing Inflammation (n = 3)
The discrepancies between results obtained by DPI and the dissection could not only be explained by biodiversity but could also be due to the well-known differences in resolution and counts acquisition between the methods used.
DISCUSSION
2-Iodo-l-phenylalanine was developed as an alternative SPECT tumor tracer to 4-iodo-l-phenylalanine, 2-iodo-l-tyrosine, and 3-iodo-α-methyltyrosine to overcome the high blood-pool and inflammation activity, the difficult precursor synthesis, and the marked long-term renal accumulation, respectively (15–17).
Cu1+-Assisted nucleophilic exchange provides not only an easy 1-step reaction for precursor synthesis but also the possibility to establish a kit formulation for fast and quantitative routine radioiodination of 2-I-l-phenylalanine without the need of an extra purification procedure.
In vitro in R1M cells, the uptake kinetics and the Michaelis–Menten constant values of 125I-2-iodo-l-phenylalanine and the natural amino acid l-phenylalanine are both similar and both amino acids are transported by the obligatory exchange LAT1 system. Moreover, our research group revealed that 125I-2-iodo-l-phenylalanine is not incorporated into the cell proteins and that the uptake of the tracer at equilibrium reflects the total “amino acid turnover” of the R1M tumor cells. Recent evidence verified that incorporation of radiolabeled amino acids is not necessary for tumor imaging and that the increased uptake is directly related to the metabolic requirement of the tumor cells (8).
With regard to these characteristics, the new tracer 123/125I-2-iodo-l-phenylalanine, which has a free para position and is somewhat less lipophilic than 4-iodo-l-phenylalanine, was evaluated in vivo in view of its potential application for oncologic imaging with SPECT.
The planar imaging experiments with the new tracer showed that the same uptake kinetics, compared with the in vitro experiments, could be applied: The uptake as a function of time followed a rectangular hyperbolic curve and Figure 3B indicated that for the uptake a pseudo reversible, pseudo first-order reaction was followed (8). These observations strongly suggest that, in vivo, 123/1225I-2-iodo-l-phenylalanine is also transported by the LAT1 system.
The latter hypothesis is confirmed by the displacement study. Although the displaced 123I-2-iodo-l-phenylalanine radioactivity in the tumor by l-phenylalanine, l-methionine, and l-cysteine was small, it was significant (Table 1). This shows that the transporter system involved is of the “obligatory exchanger” type, such as LAT1. The rather small amount of displaced radioactivity can be explained by the fact that the blood plasma is continuously filtered in the kidneys by the glomerulus and the Bowman’s capsule. When a huge excess of amino acids, such as l-phenylalanine and l-methionine, is administered to the animals, the excess will be immediately excreted to the urinary tractus.
123/125I-2-Iodo-l-phenylalanine accumulated quickly to reach high amounts in the R1M tumors, and only a very small part showed dehalogenation after 90 min (Fig. 5; Table 2). Moreover, low abdominal background, fast blood clearance, low brain uptake, and almost negligible uptake in inflammatory tissue (T/I ratios of 1.3 and 1.6), in comparison with 18F-FDG (T/I ratios of 5.9 and 8.5), were observed. These findings are also confirmed in the literature for another radiolabeled amino acid, 2-iodo-l-tyrosine, which showed a much lower uptake in the site of inflammation in comparison with 18F-FDG (17).
Just like 123I-2-iodo-l-tyrosine, 123I-4-iodo-l-phenylalanine, and 123I-3-iodo-α-methyltyrosine uptake in rodents (17,18), 125I-2-iodo-l-phenylalanine showed high uptake in the pancreas. This is typical for labeled amino acid analogs in rodents. In patients, no significant pancreatic uptake was observed for 2-iodo-l-tyrosine and 123I-3-iodo-α-methyltyrosine (15,17).
The relationship between the time to reach the steady state for 123I-2-iodo-l-phenylalanine tumor uptake in vivo and the tumor volume reached a plateau phase at a tumor volume of 4 cm3, which could be due to the higher level of necrosis in the larger tumors. It was shown that, even when the radiolabeled synthetic amino acid tumor tracer is not incorporated into cell proteins, the uptake reflects the total amino acid turnover of the tumor cells (8).
CONCLUSION
123I-2-Iodo-l-phenylalanine is a promising tracer for intracranial as well as extracranial oncologic imaging with SPECT because of its high and fast tumor accumulation, low renal and brain uptake, and minor accumulation in acute inflammatory lesions. Furthermore, its chemical synthesis is easy and allows a kit formulation, which is a benefit for transferring the tracer to clinical use.
Acknowledgments
Financial support was given by the Fonds voor Wetenschappelijk Onderzoek.
Footnotes
Received May 5, 2004; revision accepted Nov. 17, 2004.
For correspondence or reprints contact: Veerle Kersemans, MSc, Laboratory of Radiopharmacy, Harelbekestraat 72, B-9000 Gent, Belgium.
E-mail address: veerle.kersemans{at}ugent.be
