Synthesis and in vivo Evaluation of N-[N-[(S)-1,3-Dicarboxypropyl]carbamoyl]-4-[¹⁸F]fluorobenzyl-L-cysteine, [¹⁸F]DCFBC: a New Imaging Probe for Prostate Cancer

Abstract

Previously we demonstrated successful imaging of xenografts that express the prostate-specific membrane antigen (PSMA) using small animal positron emission tomography (PET) and the radiolabeled PSMA inhibitor N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-S-[¹¹C]methyl-L-cysteine, [¹¹C]DCMC. Herein we extend that work by preparing and testing a PSMA inhibitor of the same class labeled with fluorine-18 to facilitate clinical use.

Methods

N-[N-[(S)-1,3-Dicarboxypropyl]carbamoyl]-4-[¹⁸F]fluorobenzyl-L-cysteine, [¹⁸F]DCFBC was prepared by reacting 4-[¹⁸F]fluorobenzyl bromide with the corresponding thiol precursor. SCID mice bearing subcutaneous PSMA+ PC-3 PIP and PSMA- PC-3 FLU tumors were administered [¹⁸F]DCFBC via tail vein injection for ex vivo biodistribution and in vivo imaging. For biodistribution, mice were sacrificed and tumor, blood, and major organs were harvested, weighed, and radioactivity was counted. Imaging was performed using small animal PET.

Results

The radiochemical yield for [¹⁸F]DCFBC averaged 16 ± 6% (n = 8) from 4-[¹⁸F]fluorobenzyl bromide. Specific radioactivities averaged 52 GBq/μmol (1,392 Ci/mmol, n = 6). Biodistribution and imaging studies showed high uptake of [¹⁸F]DCFBC in the PIP tumors with little to no uptake in FLU tumors. The maximum PIP tumor uptake was 8.16 ± 2.55 %ID/g, achieved at 60 min after injection, which decreased to 4.69 ± 0.89 at 120 min. The PIP tumor/muscle ratio was 20 at 120 min postinjection. Based on the mouse biodistribution, the dose-limiting organ is kidney (human estimated absorbed dose: 0.05 mGy/MBq; 0.2 rad/mCi).

Conclusion

[¹⁸F]DCFBC localizes to PSMA+ expressing tumors in mice, permitting imaging by small animal PET. This new radiopharmaceutical is an attractive candidate for further studies of PET imaging of prostate cancer.

INTRODUCTION

Prostate cancer is the leading cancer in the U.S. population and second leading cause of cancer death in men (1). Staging of the disease becomes more important as new therapeutic options – such as thermal ablation or high-intensity focused ultrasound – become available. Staging can be performed noninvasively with imaging, and a number of experimental radiopharmaceuticals are under evaluation (2), (3), (4), (5). One important indication for imaging prostate cancer is to determine the location of recurrence in patients who have undergone prostatectomy who present with rising prostate-specific antigen (PSA). That is the indication for using ProstaScint™, a monoclonal antibody-based imaging agent that binds to the prostate-specific membrane antigen (PSMA) (6). We have also been interested in PSMA as an imaging target, not only to detect prostate cancer (7), (8), (9), but also because PSMA is upregulated in the neovasculature of many tumor types (10). That latter characteristic of PSMA has recently been exploited in monoclonal antibody-based imaging of solid tumors other than prostate (11).

In our effort to develop a PSMA-based imaging agent of low molecular weight for positron emission tomography (PET) with more widespread utility, i.e., a longer physical half-life and potentially better pharmacokinetic characteristics, we have extended our previous work with the carbon-11-labeled compound N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-S-[¹¹C]methyl-L-cysteine, [¹¹C]DCMC to one containing fluorine-18, N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-[¹⁸F]fluorobenzyl-L-cysteine, [¹⁸F]DCFBC. Herein we describe the synthesis, in vivo behavior and human dosimetry estimates for [¹⁸F]DCFBC.

MATERIALS AND METHODS

N^α-Fmoc-S-tert-butyl-L-cysteine (1) was purchased from AnaSpec, Inc., San Jose CA. All other reagents and solvents were purchased from either Sigma-Aldrich (Milwaukee, WI) or Fisher Scientific (Pittsburgh PA). ¹H NMR spectra were obtained on a Varian Mercury 400 mHz Spectrometer. Optical rotation was measured on a Jasco P-1010 Polarimeter. Mass spectrometry was performed on a JOEL JMS-AX505HA mass spectrometer in the Mass Spectrometry Facility at the University of Notre Dame. Melting points were measured using a Mel-Temp apparatus and are uncorrected. High performance liquid chromatography (HPLC) purification of (S)-2-[3-[(R)-1-carboxy-(2-mercapto)ethyl]ureido]pentanedioic acid (6) and N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine (DCFBC) was performed on a Waters 625 LC system with a Waters 490E multiwavelength UV/Vis detector, both controlled by Millennium v2.10 software. Reverse phase radio-HPLC analysis of 4-[¹⁸F]fluorobenzylbromide (4-[¹⁸F]FBB) was performed using a Waters 610 HPLC pump, a Waters 441 fixed wavelength (254) UV detector, a Bioscan Flow Count PIN diode radioactivity detector, a 3.9 × 150mm, 5μm Nova Pak C-18 column, and a mobile phase of 40:60 (v/v) acetonitrile:0.1M aqueous ammonium formate. Chromatograms were analyzed using a Varian Galaxie Chromatography Data System Version 1.8.501.1. Reverse phase radio-HPLC semipreparative purification of N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-[¹⁸F]fluorobenzyl-L-cysteine, [¹⁸F]DCFBC, was performed using a Waters 510 pump, Waters 490E variable wavelength UV/Vis detector at 220 nm, a Bioscan Flow Count PMT radioactivity detector, a 10 mm × 250 mm 10 μ Alltech Econosil C-18 column, and WinFlow (LabLogic) chromatography software. [¹⁸F]NaF was produced by 18MeV proton bombardment of a high pressure [¹⁸O]H₂O target using a General Electric PETtrace biomedical cyclotron (Milwaukee, WI). Radioactivity was measured in Capintec 15R and CRC-12 dose calibrators. The specific radioactivity was calculated as the radioactivity eluting at the retention time of [¹⁸F]DCFBC during the semi-preparative HPLC purification divided by the mass corresponding to the area under the curve of the UV absorption.

Chemistry

N^α-Fmoc-S-tert-butyl-L-cysteine-4-methoxybenzyl ester (2)

To a solution of 4.0 g (10 mmol) of N^α-Fmoc-S-tert-butyl-L-cysteine (1) dissolved in 80 mL of dry DMF was added 6.5 g (20 mmol) of cesium carbonate and 2.4 g (15.3 mmol) of 4-methoxybenzyl chloride. The suspension was stirred at room temperature under nitrogen for four hours. The suspension was filtered through a medium frit Büchner funnel and the solid washed with CH₂Cl₂. The DMF and CH₂Cl₂ washes were combined and poured into 200 mL of ethyl acetate and extracted with 150 mL of water. The organic layer was washed twice with 150 mL of water, collected, dried over anhydrous magnesium sulfate, filtered, and concentrated on the rotary evaporator to give a colorless oil that solidified overnight. The solid was then recrystalized from 7:3 (v/v) hexane:ethylacetate to give 3.4 g (65.5 % yield) of a white solid (mp 117–119° C). The filtrate was concentrated, dissolved in 2 mL of CH₂Cl₂, loaded onto a silica gel column and eluted with 8:2 hexane:ethyl acetate to give an additional 0.7 g (13 %) of product. TLC: silica gel on Al backing, 7:3 hexanes:ethyl acetate, R_f = 0.66. ¹H NMR (CDCl₃) δ 7.77 (d, 2H, J = 6 Hz), 7.60 (d, 2H, J = 6 Hz), 7.40 (t, 2H, J = 7.5 Hz), 7.31 (m, 4H), 6.87 (d, 2H, J = 6.75), 5.64 (d, 1H, J = 7.5 Hz), 5.14 (d, 2H, J = 3 Hz), 4.68 (m, 1H), 4.37 (m, 2H), 4.22 (t, 1H, J = 7.5 Hz), 3.8 (s, 3H), 3.01 (d, 2H, J = 5 Hz), 1.28 (s, 9H). [α]²⁴_D (c 0.106, DMF) = −11.5; FAB+ m/z 520.2135 [MH⁺].

S-tert-butyl-L-cysteine-4-methoxybenzyl ester (3)

Thirty milliliters of a 20 % solution of piperidine in DMF was added to 2.6 g (5 mmol) of N^α-Fmoc-S-tert-butyl-L-cysteine-4-methoxybenzyl ester (2) in a flame dried round bottom flask. The reaction was stirred at room temperature for 10–15 min, poured into a separatory funnel containing 100 mL of CH₂Cl₂. This was extracted three times with 50 mL of water followed by 100 mL of saturated NaCl. The organic layer was collected, dried over MgSO₄, filtered and concentrated to a sticky white solid. The crude material was purified by flash column chromatography using silica gel and 1:1 hexanes:ethyl acetate to give 1.26 g (85 %) of a light yellow oil. TLC: 1:1hexanes:ethyl acetate, R_f = 0.38. ¹H NMR (CDCl₃) δ7.25 (d, 2H, J = 7 Hz), 6.84 (d, 2H, J = 7 Hz), 5.10 (d, 2H, J = 3 Hz), 3.75 (s, 3H), 3.62 (dd, 1H J = 3, 5 Hz), 2.88 (dd, 1H, J = 3.5, 9.7 Hz), 2.72 (dd, 1H, J = 5.5, 9.7 Hz), 1.72 (broad s, 2H), 1.26 (s, 9H).[α]²⁴_D (c 0.0318, DMF) = +3.4.

Bis-4-methoxybenzylglutamate hydrochloride (4)

Bis-4-methoxybenzylglutamate hydrochloride was prepared by the method of Maclaren (12) as a white solid. mp 120–121° C, Lit. 114–115° C (12). ¹H NMR (CDCl₃) δ7.23 (d, 2H, J = 6.4 Hz), 7.18 (d, 2H, J = 6.4Hz), 6.81 (d, 2H, J = 2.8 Hz), 6.78 (d, 2H, J = 2.8 Hz), 5.06 m, 2H), 4.93 (s, 2H), 4.29 (t, 1H, J = 5.2 Hz), 3.75 (s, 3H), 3.72 (s, 3H), 2.65 (m, 1H), 2.54 (m, 1H), 2.37 (m, 2H). [α]²⁵_D (c 0.195, DMF) = +3.1, Lit. (12) [α]²⁰_D (c 2, DMF) = +5.3.

2-{3-[2-tert-butylsulfanyl-1-(4-methoxy-benzyloxycarbonyl)-ethyl]-ureido}-pentanedioic acid bis-(4-methoxybenzyl) ester (5)

A dry 250 mL round-bottomed flask was charged with 3.28 g (7.76 mmol) of bis-4-methoxybenzylglutamate hydrochloride (4) and dissolved in 45 mL of dry CH₂Cl₂, followed by cooling to −77°C under a nitrogen atmosphere. A solution of 0.77 g of triphosgene (2.59 mmol) dissolved in 8 mL of dry CH₂Cl₂ was added. A solution consisting of 2.3 mL of triethylamine (16.5 mmol) in 8 mL of dry CH₂Cl₂ was carefully added and the reaction was allowed to stir at −77°C for 1.5 h, before warming to room temperature over 15 min. To this was added a solution containing 2.1 g (7.05 mmol) S-tert-butyl-L-cysteine-4-methoxybenzyl ester (3) dissolved in 13 mL of dry CH₂Cl₂. The mixture stirred overnight at room temperature, followed by extraction with CH₂Cl₂, two washes with water and a wash with brine. The organic layer was collected, dried over Na₂SO₄, filtered and concentrated to a thick oil. The crude material was purified on a silica gel column using 10:1 chloroform:ethyl acetate to give 3.75 g of a thick oil (75 % yield from 4). TLC: silica gel on Al backing, 10:1 chloroform/ethyl acetate, R_f = 0.49. ¹H NMR (CDCl₃) δ7.28–7.22 (m, 6H), 6.88–6.84 (m, 6H), 5.9–5.6 (broad S, 2H), 5.08 (d, 4H, J = 3Hz), 5.0 (s, 2H), 4.76 (t, 1H, J = 4.5), 4.52 (dd, 1H, J = 4.5, 6Hz), 3.79 (s, 9H), 2.95 (t, 2H, J = 4.5), 2.38 (m, 2H), 2.15 (m, 1H), 1.95 (m, 1H), 1.23 (s, 9H). [α]²⁵_D (c 0.081, DMF) = −1.14.

(S)-2-[3-[(R)-1-carboxy-(2-mercapto)ethyl]ureido]pentanedioic acid (6)

A 0 °C solution containing 15 mL of trifluoroacetic acid and 0.3 mL of anisole was added to a RBF containing 0.815 g (1.16 mmol) of 5, and the mixture stirred until 5 dissolved. To this solution was added 0.453 g (1.42 mmol) of mercuric acetate. The mixture was stirred at 0 °C for 20 min followed by concentration at room temperature under reduced pressure. The mercury adduct was precipitated by the addition of ethyl ether, filtered and then dried under reduced pressure to give 0.69 g of a fine white solid. This was used without further purification. The mercury adduct (0.69 g) was dissolved in 25 mL of DMF and H₂S was bubbled into this solution for 5 h. The resulting black slurry was filtered through filter agent (Celatom FW-14) and the filter agent washed with methanol and water. Following concentration under reduced pressure, the product was then dissolved in methanol and filtered over glass paper, the filtrate concentrated and the process repeated using water. The filtrate was then purified by RP-HPLC (C18 Econosil 10 × 250 mm preparative column; 95:5:0.1 H₂O:CH₃CN:TFA) to give 0.274 g (80 %) of a light yellow colored hygroscopic solid. ¹H-NMR (CD₃OD) δ 4.53 (t, J = 5Hz, 1H), 4.31 (dd, J = 5, 10Hz, 1H), 2.93 (d, J = 4Hz, 2H), 2.42 (m, 2H), 2.15 (m, 1H), 1.90 (m, 1H). Lit. ¹H-NMR (CD₃OD) δ4.45 (t, J = 4.5Hz, 1H), 4.21 (dd, J = 5, 8.5Hz, 1H), 2.82 (d, J = 4.5Hz, 2H), 2.31 (m, 2H), 2.05 (m, 1H), 1.80 (m, 1H); [α]²⁴_D +13.3 (c 0.0247, MeOH), Lit: (13) [α]²⁰_D +14.2 (c 0.12, MeOH); FAB+ m/z 295.0606 [MH⁺].

N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine, DCFBC

(S)-2-[3-[(R)-1-carboxy-(4-fluorobenzylsulfanyl)ethyl]ureido]pentanedioic acid (6) (1.8 mg, 0.0061 mmol) and 4-fluorobenzyliodide (1.4 mg, 0.0061 mmol) were stirred in ammonia-saturated methanol (0.3 mL) at 60 °C for 10 min then acidified with TFA. Purification by RP-HPLC, C18 Econosil 10 × 250 mm preparative column; (32:68:0.1 CH₃CN:H₂O:TFA) gave pure product. ¹H NMR (CD₃OD) δ 7.35 (dd, 2H), 7.01 (dd, 2H), 4.49 (m, 1H), 4.32 (m, 1H), 3.78 (s, 2H), 2.84 (m, 2H), 2.42 (m, 2H), 2.15 (m, 1H), 1.92 (m, 1H). FAB m/z 401.0845 [M+H].

Radochemistry

N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-[¹⁸F]fluorobenzyl-L-cysteine, [¹⁸F]DCFBC

Typically, starting with 12.95 GBq (350 mCi) of [¹⁸F]NaF, a solution containing 91 % 4-[¹⁸F]FBB (14) was produced in 45 min (total reactivity 3.4 GBq (93 mCi)) and placed in a 5 mL screw-cap borosilicate glass vial. To this solution (1.5 mL), still containing the brominating agent triphenylphosphine dibromide in methylene chloride/ether, was added two 4 mL volumes of diethyl ether to precipitate the triphenylphosphine dibromide. After standing for 1 min, the liquid phase was carefully transferred via a plastic pipette to a 16 × 150 mm borosilicate glass vial to which 800 μL methanol had been added previously. The liquid was concentrated under a stream of nitrogen at room temperature to approximately 200 μL. This solution was then added to a 4 mL borosilicate screw-cap glass vial containing an aqueous solution of (S)-2-[3-[(R)-1-carboxy-(2-mercapto)ethyl]ureido]pentanedioic acid (6) (2–4 mg/40 μL). To this was added 200 μL of methanol previously saturated with ammonia gas. The vial was sealed and heated at 65 °C for 10 min, then cooled for two minutes. To the reaction was added 600 μL water, 80 μL of trifluoroacetic acid (a 5 μL sample was spotted on pH paper to confirm acidity), and 600 μL of HPLC mobile phase. The resulting product was then purified by reverse phase semi-preparative radio-HPLC using a mobile phase consisting of 35:65:0.1 % acetonitrile:water:trifluoroacetic acid and a flow rate of 4 mL/min, yielding 490 MBq (13.2 mCi) of [¹⁸F]DCFBC eluting at 7 min. The product was concentrated under vacuum to dryness, reconstituted in 1 mL of phosphate-buffered saline (pH = 7.4), and filtered through a 0.22 μm syringe filter into an evacuated sterile vial.

In Vitro Inhibition Assay

Taking advantage of the N-acetylaspartylglutamate (NAAG) peptidase activity of PSMA, the relative affinity of DCFBC for PSMA was determined using a previously published NAAG peptidase assay (15). Briefly, NAAG peptidase activity was determined using membranes of CHO cells stably transfected with NAAG peptidase (a.k.a. PSMA), 4 μM NAAG as a substrate and a trace amount of [³H]NAAG. Inhibitors at concentrations of 0.1, 1, 10 and 100 nM were tested. Product was separated using ion exchange chromatography (AG-50W-X8 analytical grade cation-exchange resin). The amount of [³H]glutamate as a product of NAAG hydrolysis was determined by scintillation spectrophotometry.

Cell Lines and Mouse Models

PC-3 PIP (PSMA+) and PC-3 FLU (PSMA−) cell lines were obtained from Dr. Warren Heston (Cleveland Clinic) and were maintained as previously described (16). All cells were grown to 80−90% confluency before trypsinization and formulation in Hank's Balanced Salt Solution (HBSS, Sigma) for implantation into mice.

All animal studies were carried out in full compliance with institutional guidelines related to the conduct of animal experiments. Male SCID mice (Charles River Laboratories, Wilmington, MA) were implanted subcutaneously with 1–5 × 10⁶ cells forward of each shoulder. PC-3 PIP cells were implanted behind the left shoulder and PC-3 FLU cells were implanted behind the right shoulder. Mice were imaged or used in biodistribution assays when the tumor xenografts reached 3–5 mm in diameter.

Rodent Biodistribution

The xenograft-bearing mice (17–20 g) were injected via the tail vein with 3.70 MBq (100 μCi, 286 pmol, 350 Ci/mmol) of [¹⁸F]DCFBC in 200 μL of saline. Blood was collected immediately after sacrifice (cervical dislocation) by cardiac puncture and heart, lung, liver, stomach, pancreas, spleen, white fat, kidney, bone, muscle, small intestine, large intestine, urinary bladder, tumor xenografts, cerebral cortex and cerebellum were harvested, weighed and counted in an automated gamma counter (LKB Wallace 1282 Compugamma CS Universal Gamma Counter). Animals were sacrificed at 5, 15, 30, 60 and 120 min post-injection (n = 4 per time point). Tissue radiopharmaceutical uptake values were calculated as percent injected dose per gram (% ID/g) as compared with a 1:10 diluted standard dose. The urinary bladder was emptied and water washed and then dried prior to weighing and counting.

Small Animal PET

A SCID mouse bearing subcutaneous PC-3 PIP and PC-3 FLU xenografts was anesthetized using 3% isoflurane in oxygen for induction and 1.5% isoflurane in oxygen at 0.8 L/min flow for maintenance and positioned prone on the gantry of a GE eXplore Vista small animal PET scanner (GE Healthcare, Milwaukee, WI). The mouse was injected intravenously with 7.4 MBq (200 μCi, 572 pmol, 350 Ci/mmol) of [¹⁸F]DCFBC followed by image acquisition using the following protocol: The images were acquired as a pseudodynamic scan, i.e., a sequence of successive whole-body images acquired in two bed positions for a total of two hours. The dwell time at each position was 5 minutes, such that a given bed position (or mouse organ) was revisited every 10 min. An energy window of 250–700 keV was used. Images were reconstructed using the FORE/2D-OSEM method (2 iterations, 16 subsets) and included corrections for radioactive decay, scanner dead time and scattered radiation.

Human Dosimetry Estimates

Dosimetry values were calculated using mouse biodistribution data. The mouse activity values in %ID/g where converted to human %ID/organ by setting the ratio of organ %ID/g to whole-body %ID/g in the mouse equal to that in humans and then solving for human %ID/organ; the adult male organ masses listed in the software code OLINDA were used (17). Cumulative activity per injected activity for each organ was obtained by numerically integrating over the existing time points, then fitting a mono-exponential curve to the last 3 time points to extrapolate effective clearance rate beyond the last measured time point. The sum of the numerical (measured data), and the analytical integrals gave the total cumulated activity. This was divided by the amount of activity administered to give cumulated activity per unit activity. The resulting residence times in MBq-h/MBq, were entered into OLINDA to calculate absorbed and effective doses.

Since DCFBC is a small molecule whose concentration in blood is not readily related to that in marrow, the residence time in blood was assigned along with adipose tissue (fat) to the remainder body term and not apportioned to the red marrow. Values for residence times in the lower large intestine, small intestine, stomach, upper large intestine, and urinary bladder all reflect the contents as well as the tissue of the indicated organs.

RESULTS

Chemistry and PSMA Inhibition

Scheme 1 depicts the synthesis of DCFBC and [¹⁸F]DCFBC. The synthesis of 6 is a modification of the route previously described by Kozikowski et al., (13) where the benzyl protecting groups are now replaced by the acid labile p-methoxybenzyl groups. In particular, the carboxyl group of commercial N^α-Fmoc-S-tertbutyl-L-cysteine (1) is protected as a p-methoxybenzyl ester to give 2. The Fmoc group is removed, giving amine 3, which is then reacted with the isocyanate formed from bis-4-methyoxybenzyl glutamate hydrochloride (4) (12) to give urea 5. Cleavage of all remaining protecting groups gives precursor 6.

The fluorine-18-labeled prosthetic group 4-[¹⁸F]FBB was prepared by the method of Ravert et al. (14) and reacted with 6 in ammonium-saturated methanol at 60 °C for 10 min, followed by acidification and purification by reverse phase radio-HPLC. The average uncorrected yield of [¹⁸F]DCFBC from [¹⁸F]FBB was 16 ± 6% (n = 8). In a typical experiment, the non-decay corrected yield of the total synthesis from [¹⁸F]NaF was 3.5 % in 123 min (decay-corrected yield was 7.6 %). The specific radioactivites ranged from 13–133 GBq/μmol (350–3,600 Ci/mmol) with an average of 52 GBq/μmol (1,492 Ci/mmol, n = 6).

A NAAG peptidase inhibition assay (15) was undertaken to determine the IC₅₀ value for DCFBC and thus its inhibitory capacity for PSMA. The concentration of DCFBC was varied from 1 nM to 100 nM against a fixed amount of NAAG (4 μM) and a trace amount of [³H]NAAG. The NAAG peptidase (PSMA) was prepared from lysed PSMA-transfected CHO cells. The percent enzymatic cleavage product, [³H]glutamate, was measured by scintillation counting and plotted against the logarithmic concentration of DCFBC. Linear regression of the resulting data were solved for 50% [³H]glutamate (50% inhibition) and resulted in an IC₅₀ value of 13.9 nM for DCFBC. That result is in keeping with other compounds of this class (13), (7), (8).

Rodent Biodistribution and PET Imaging

Table 1 outlines the ex vivo rodent tissue distribution results. The blood, kidney, urinary bladder, spleen and PSMA+ PC-3 PIP tumor display high uptake at the initial, five min postinjection (p.i.) time point. By 60 min p.i., the kidneys and urinary bladder display the highest uptake while the uptake in PSMA+ PC-3 PIP tumor achieves its highest absolute value. The values noted in the kidney are largely due to specific binding rather than renal clearance, due to the expression of high amounts of PSMA in the proximal renal tubule (18), (19). Urinary bladder uptake represents excretion at all time points, i.e., there was no specific binding to bladder wall, while tumor uptake demonstrates a high degree of specificity represented by the PIP:FLU uptake ratio of 10:1 at 60 min and rising to 20:1 at 120 min (Table 2). Tumor to other organ ratios also increase with time.

Table 1.

Tissue distribution of [¹⁸F]DCFBC

	%ID/g ± SD (n = 4)
tissue	5 min	15 min	30 min	60 min	120 mina
blood	11.3 ± 6.7	4.1 ± 2.5	2.3 ± 1.3	1.8 ± 1.4	0.4 ± 0.2
heart	4.4 ± 0.9	2.0 ± 0.6	1.2 ± 0.2	0.8 ± 0.3	0.3 ± 0.2
lung	7.0 ± 0.5	3.2 ± 0.9	1.8 ± 0.3	1.1 ± 0.4	0.4 ± 0.1
liver	6.0 ± 1.2	4.1 ± 1.4	4.2 ± 0.5	5.1 ± 0.8	2.1 ± 1.4
stomach	2.9 ± 0.3	1.4 ± 0.3	0.8 ± 0.1	0.5 ± 0.2	1.1 ± 1.9
spleen	8.8 ± 1.3	4.3 ± 1.4	1.9 ± 0.9	1.6 ± 0.9	0.4 ± 0.2
fat	1.8 ± 0.4	1.6 ± 1.0	0.7 ± 0.5	1.0 ± 0.7	0.3 ± 0.1
kidney	63.4 ± 8.2	63 ± 20	51.3 ± 7.5	41.6 ± 7.2	13 ± 10
bone	2.9 ± 0.4	1.6 ± 0.3	1.7 ± 0.1	1.7 ± 0.8	2.5 ± 3.6
muscle	2.2 ± 1.0	1.0 ± 0.4	0.5 ± 0.1	0.6 ± 0.5	0.2 ± 0.4
sm. intest.	4.3 ± 0.4	2.3 ± 1.3	1.2 ± 0.2	0.7 ± 0.2	0.2 ± 0.1
lrg. intest.	2.8 ± 0.4	1.5 ± 0.7	0.8 ± 0.1	0.6 ± 0.2	0.3 ± 0.1
bladder (empty)	55 ± 30	16 ± 15	15 ± 15	14.5 ± 7.3	2.6 ± 1.0
PC-3 FLU tumor	3.5 ± 0.5	1.7 ± 0.4	1.0 ± 0.1	0.8 ± 0.2	0.2 ± 0.0
PC-3 PIP tumor	8.2 ± 0.8	6.0 ± 2.6	6.2 ± 1.0	8.2 ± 2.5	4.7 ± 0.9

^an = 3

Table 2.

PC-3 PIP tumor/tissue ratios

Tissue	5 min	15 min	30 min	60 min	120 mina
blood	0.7	1.5	2.7	4.5	13
heart	1.9	3.0	5.0	10	17
lung	1.2	1.9	3.5	7.4	30
liver	1.4	1.5	1.5	1.6	2.2
stomach	2.8	4.5	7.6	18	4.3
spleen	0.9	1.4	3.2	5.2	11
fat	4.7	3.9	9.2	7.8	18
kidney	0.1	0.1	0.1	0.2	0.4
bone	2.8	3.8	3.6	4.7	1.9
muscle	3.7	6.0	14	14	20
sm. intest.	1.9	2.6	5.1	12	31
lrg. intest.	2.9	4.1	7.9	14	17
bladder (empty)	0.15	0.4	0.4	0.6	1.8
FLU tumor	2.4	3.5	6.4	11	27

^an = 3

Figure 1 shows the PET scans at specific time intervals. Specific uptake in PSMA+ PIP tumor is clearly seen as early as the 20–30 min image. Clearance from nontarget tissues is evident at later time points. By two hours radioactivity in the kidney is confined to cortex. The renal activity steadily decreased throughout the time course investigated and did so more rapidly than in other tissues, including the PIP tumors (Figure 2). Little nonspecific tissue radioactivity uptake is evident. Renal excretion dominates with this hydrophilic compound.

Figure 1.

Figure 2.

Human Dosimetry Estimates

Table 3 indicates human dosimetry estimates for [¹⁸F]DCFBC. Tables 3 and 4 list the absorbed doses and residence times, respectively, estimated from the mouse biodistribution data. The highest absorbed dose was to the kidneys (0.05 mGy/MBq (0.2 rad/mCi)). Excluding the kidneys, mean absorbed doses ranged from 0.009 (liver) to 0.0005 (brain) mGy/MBq (0.035–0.002 rad/mCi). The effective dose equivalent and effective doses were 0.005 and 0.003 mSv/MBq, respectively.

Table 3.

Radiation absorbed doses (AD)^*

	Total AD (mGy/MBq)
Brain	4.97E-04
LLI Wall	1.01E-03
Small Intestine	1.45E-03
Stomach Wall	1.57E-03
ULI Wall	1.53E-03
Heart Wall	2.25E-03
Kidneys	4.87E-02
Liver	9.34E-03
Lungs	2.58E-03
Muscle	1.37E-03
Pancreas	3.03E-03
Red Marrow*	3.12E-03
Spleen	4.25E-03
Urinary Bladder Wall	8.99E-04
Total Body	1.79E-03

^*Estimate reflects dose contribution from other organs. Absorbed dose values in mGy/MBq per organ expected in average human.

Table 4.

Radiation residence times*

Brain	8.76E-04
LLI	1.01E-03
Small Intestine	4.00E-03
Stomach	6.20E-04
ULI	1.01E-03
Heart Wall	1.94E-03
Kidneys	7.85E-02
Liver	6.78E-02
Lungs	9.39E-03
Muscle	8.41 E-02
Pancreas	3.66E-04
Trabecular Bone	5.24E-02
Spleen	2.19E-03
Urinary Bladder Contents	3.98E-03
Remainder	7.37E-02

The residence times obtained for each organ in an average human reported in MBq-h/MBq.

DISCUSSION

Prostate cancer is the most common solid tumor in men (20). Testing for PSA in serum can suggest the presence or recurrence of tumor, but provides no spatial information, which dictates therapy. While it is true that nomograms, comprised of clinical parameters such as PSA level and velocity, have proved useful in predicting local extension, they do less well in predicting lymph node involvement (21). In fact lymph node involvement is often underestimated in prostate cancer and detection of such involvement – or involvement of other metastatic sites – could obviate unnecessary surgery (21), (22). One study found that 4.5% of patients who underwent abdominoperineal resection for colorectal cancer had perirectal lymph nodes that contained prostate cancer (23). In brief, to predict outcome, provide image-guided therapy and facilitate therapeutic monitoring, an imaging agent capable of detecting prostate cancer with high specificity and sensitivity is needed. As discussed in the introduction, certain mechanism-based agents are under investigation, including a magnetic resonance imaging-based agent that has tremendous promise in detecting nodal disease (24). However, only ProstaScint™ has gained FDA approval. ProstaScint™ has not been as widely implemented as hoped, primarily due to suboptimal pharmacokinetics characteristic of antibody-based imaging agents. High resolution SPECT devices coupled with fused (SPECT-CT) imaging has improved the detection of metastatic deposits with ProstaScint™, however, accuracy remains at 83%, leaving room for improvement (21). Furthermore, results obtained with ProstaScint™ have been controversial, with some reports claiming no advantage in using the scan in patients who are postprostatectomy with rising PSA (25).

Like ProstaScint™, [¹⁸F]DCFBC binds to PSMA, the most well established and highly restricted prostate cancer-related membrane antigen (26). PSMA is upregulated in prostate cancer, particularly in advanced, hormone-independent and metastatic disease (27), (28). It is also expressed in the neovasculature of nearly all solid tumors (10), (11). Because it is an integral membrane protein with an enzymatic active site in an extracellular domain, it provides an ideal target for imaging and therapy. Adding further to the attractiveness of PSMA as an imaging target is its limited pattern of expression, primarily within prostate, small bowel, proximal renal tubule and brain (19). Within the brain PSMA is known as glutamate carboxypeptidase II (GCPII, a.k.a. NAAG peptidase), where it catalyzes the hydrolysis of NAAG to glutamate and N-acetylaspartate (18), (29). PSMA/GCPII is an active target for the development of imaging agents for prostate cancer, and therapeutic agents for psychiatric disease, with several reviews having recently appeared (30), (31), (32), (33). There is an emerging literature on the development of inhibitors for PSMA/GCPII, which is expected to increase with the recent availability of a high-resolution crystal structure of the enzyme (34), (35), (13), (36). Others and we have synthesized radiopharmaceuticals and optical agents for PSMA detection (37), (38), (9), (7), (8), with [¹⁸F]DCFBC representing the first agent designed specifically for clinical PET imaging of prostate cancer.

[¹⁸F]DCFBC localized selectively to the PSMA+ prostate tumor, PC3-PIP (Figure 1, Tables 1 and 2), achieving a target:background (muscle) of 20:1 at 120 min postinjection. The time-activity curves indicate that [¹⁸F]DCFBC has achieved equilibrium by 120 min and has begun to decrease in concentration at the target site. Washout from target was slower than from nontarget sites. Notably, the criteria for ProstaScint™ to progress to the clinic required a 3:1 tumor:muscle in subcutaneous LNCaP tumors, and a similar uptake ratio demonstrated in the clinic provided a positive predictive value for the presence of prostate cancer at > 90% (39). [¹⁸F]DCFBC did not demonstrate significant defluorination, as evidenced by the low levels of bone uptake. That is particularly important because of the propensity prostate cancer has for metastasizing to bone. The 1 hr PIP:bone value was 4.7 suggesting that metastatic lesions, which are known to express PSMA, will be clearly delineated (40), (11).

As seen for our initial PET radioligand, [¹¹C]DCMC (7), there is significant uptake within kidney that is primarily due to specific binding to proximal renal tubules. We have demonstrated this with other agents that we have developed for SPECT imaging of PSMA (data not shown). Because of the presumed high renal exposure we undertook dosimetry studies that indicate an exposure of 0.0487 mGy/MBq, which is within accepted levels (Table 3). The estimated absorbed doses resulting from expected diagnostic administration of 185 to 370 MBq (5 to 10 mCi) [¹⁸F]DCFBC are acceptable for nuclear medicine diagnostic procedures (Table 3). Vallabhajosula, et al. (41) have reported on the dosimetry of the anti-PSMA antibody, J591, labeled with indium-111. Using imaging and serial blood data obtained from a phase I study, they found that liver received the greatest absorbed dose at 1.1 mGy/MBq, the kidneys received the third greatest absorbed dose at 0.7 mGy/MBq. The absorbed doses for [¹⁸F]DCFBC are one to two orders of magnitude below the values reported for [¹¹¹In]J591.

In summary, we have synthesized the first clinically practical PET imaging agent for PSMA. [¹⁸F]DCFBC has a suitable physical half-life, appropriate pharmacokinetics and dosimetry and can be synthesized with relative ease in reasonable radiochemical yield and specific radioactivity. Together with the development of standard operating procedures for radiosynthesis according to good manufacturing practice, toxicity studies are under way en route to clinical implementation.