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`
`Imaging, Diagnosis, Prognosis
`
`N-[N-[(S)-1,3-Dicarboxypropyl]Carbamoyl]-4-[18F]Fluorobenzyl-L-
`Cysteine, [18F]DCFBC: A New Imaging Probe for Prostate Cancer
`Ronnie C. Mease,1Crystal L. Dusich,1Catherine A. Foss,1Hayden T. Ravert,1 Robert F. Dannals,1
`Jurgen Seidel,1Andrew Prideaux,1James J. Fox,1George Sgouros,1
`Alan P. Kozikowski,2 and Martin G. Pomper1
`
`Abstract Purpose: Previously, we showed 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. Herein, we extend that work by preparing and testing a PSMA inhibitor
`of the same class labeled with fluorine-18.
`Experimental Design: N-[N-[(S)-1,3-Dicarboxypropyl]carbamoyl]-4-[18F]fluorobenzyl-L-
`cysteine ([18F]DCFBC) was prepared by reacting 4-[18F]fluorobenzyl bromide with the precur-
`sor (S)-2-[3-[(R)-1-carboxy-2-mercaptoethyl]ureido]-pentanedioic acid in ammonia-saturated
`methanol at 60jC for 10 min followed by purification using C-18 reverse-phase semipreparative
`high-performance liquid chromatography. Severe combined immunodeficient mice bearing a s.c.
`PSMA+ PC-3 PIP tumor behind one shoulder and a PSMA- PC-3 FLU tumor behind the other
`shoulder were injected via the tail vein with either 1.85 MBq (50 ACi) of [18F]DCFBC for ex vivo
`biodistribution or 7.4 MBq (200 ACi) for imaging. For biodistribution, mice were sacrificed at
`5, 15, 30, 60, and 120 min. Tumor, blood, and major organs were harvested and weighed, and
`radioactivity was counted. Imaging was done on the GE eXplore Vista small-animal PET scanner
`by collecting 12 consecutive 10-min frames.
`Results: Radiochemical yield for [18F]DCFBC averaged 16 F 6% (n = 8) from 4-[18F]fluoroben-
`zyl bromide. Specific radioactivities ranged from 13 to 133 GBq/Amol (350-3,600 Ci/mmol) with
`an average of 52 GBq/Amol (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. High radio-
`pharmaceutical uptake was also seen in kidneys and bladder; however, washout of radioactivity
`from these organs was faster than from the PIP tumors. The maximum PIP tumor uptake was
`8.16 F 2.55% injected dose per gram, achieved at 60 min after injection, which decreased to
`4.69 F 0.89 at 120 min. The PIP tumor to muscle ratio was 20 at 120 min after injection. Based
`on the mouse biodistribution, the dose-limiting organ is the kidneys (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.
`
`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,
`
`Authors’ Affiliations: 1Russell H. Morgan Department of Radiology, Johns
`Hopkins Medical Institutions, Baltimore, Maryland and 2Department of Medicinal
`Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois
`Received 6/19/07; revised 8/13/07; accepted 9/28/07.
`Grant support: U24 CA92871 R21 EB005324, R21 CA111982, PC050825,
`AdMeTech and P50 103175.
`The costs of publication of this article were defrayed in part by the payment of page
`charges. This article must therefore be hereby marked advertisement in accordance
`with 18 U.S.C. Section 1734 solely to indicate this fact.
`Requests for reprints: Martin G. Pomper, Johns Hopkins Medical Institutions,
`1550 Orleans Street, 492 CRB II, Baltimore, MD 21231. Phone: 410-955-2789;
`Fax: 443-817-0990; E-mail: mpomper@jhmi.edu.
`F 2008 American Association for Cancer Research.
`doi:10.1158/1078-0432.CCR-07-1517
`
`become available. Staging can be done noninvasively with
`imaging, and several experimental radiopharmaceuticals are
`under evaluation (2 – 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; ref. 6). We have also been interested in PSMA as an
`imaging target not only to detect prostate cancer (7 – 9) but also
`because PSMA is up-regulated 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
`
`Clin Cancer Res 2008;14(10) May 15, 2008
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`[18F]DCFBC Synthesis and In vivo Evaluation
`
`S-tert-butyl-L-cysteine-4-methoxybenzyl ester (3). Thirty milliliters of
`a 20% solution of piperidine in DMF were added to 2.6 g (5 mmol) of
`Na-Fmoc-S-tert-butyl-L-cysteine-4-methoxybenzyl ester (2)
`in a flame-
`dried round-bottomed flask. The reaction was stirred at room
`temperature for 10 to 15 min and poured into a separatory funnel
`containing 100 mL of CH2Cl2. This was extracted thrice 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 hexane/ethyl acetate to give 1.26 g (85%) of a
`light yellow oil. TLC: 1:1 hexane/ethyl acetate, R f = 0.38. 1H NMR
`(CDCl3) y 7.25 (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). [a]24
`D (c 0.0318, DMF) = +3.4.
`Bis-4-methoxybenzylglutamate hydrochloride (4 ). Bis-4-methoxy-
`benzylglutamate hydrochloride was prepared by the method of
`Maclaren (12) as a white solid (mp, 120-121jC; Lit., 114-115jC;
`ref. 12). 1H NMR (CDCl3) y 7.23 (d, 2H, J = 6.4 Hz), 7.18 (d, 2H,
`J = 6.4 Hz), 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). [a]25
`D (c 0.195,
`DMF) = +3.1, Lit. (12) [a]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 -77jC 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 -77jC 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 was 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 aluminum backing, 10:1 chloroform/ethyl
`acetate, R f = 0.49. 1H NMR (CDCl3) y 7.28 to 7.22 (m, 6H), 6.88 to
`6.84 (m, 6H), 5.9 to 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). [a]25
`D (c 0.081, DMF) = -1.14.
`(S )-2-[3 -[(R )-1-carboxy-2-mercaptoethyl ]ureido ]-pentanedioic acid
`(6 ). A 0jC solution containing 15 mL of
`trifluoroacetic acid
`(TFA) and 0.3 mL of anisole was added to a round-bottom flask
`containing 0.815 g (1.16 mmol) of 5, and the mixture was stirred until
`5 dissolved. To this solution was added 0.453 g (1.42 mmol) of
`mercuric acetate. The mixture was stirred at 0jC 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 was washed with methanol
`and water. Following concentration under reduced pressure,
`the
`product was then dissolved in methanol and filtered over glass paper,
`the filtrate was concentrated, and the process was repeated using water.
`The filtrate was then purified by reverse-phase HPLC (C-18 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) y 4.53 (t, J = 5 Hz, 1H), 4.31 (dd, J = 5, 10 Hz, 1H), 2.93 (d, J
`= 4 Hz, 2H), 2.42 (m, 2H), 2.15 (m, 1H), 1.90 (m, 1H). Lit. 1H NMR
`
`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
`
`Na-Fmoc-S-tert-butyl-L-cysteine (1) was purchased from AnaSpec,
`Inc. All other reagents and solvents were purchased from either Sigma-
`Aldrich or Fisher Scientific. 1H nuclear magnetic resonance (NMR)
`spectra were obtained on a Varian Mercury 400 MHz spectrometer.
`Optical rotation was measured on a Jasco P-1010 polarimeter. Mass
`spectrometry was done 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-mercaptoethyl]ureido]-pentanedioic acid (6) and
`N -[N -[(S )-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine
`(DCFBC) were done 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]fluorobenzyl-
`bromide (4-[18F]FBB) was done 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  150 mm 5 Am Nova Pak C-18
`column, and a mobile phase of 40:60 (v/v) acetonitrile/0.1 mol/L
`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 [18F]DCFBC
`was done 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 A Alltech Econosil C-18 column, and
`WinFlow (LabLogic) chromatography software. [18F]KF was produced
`by 18 MeV proton bombardment of a high-pressure [18O]H2O target
`using a General Electric PETtrace biomedical cyclotron. 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 semipreparative HPLC
`purification divided by the mass corresponding to the area under the
`curve of the UV absorption.
`
`Chemistry
`NA-Fmoc-S-tert-butyl-L-cysteine-4-methoxybenzyl ester (2). To a solu-
`tion of 4.0 g (10 mmol) of N a-Fmoc-S-tert -butyl-L-cysteine
`(1) dissolved in 80 mL of dry N, N-dimethylformamide (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 4 h. The suspension was filtered
`through a medium frit Bu¨ chner funnel and the solid was 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/ethyl acetate to give 3.4 g
`(65.5% yield) of a white solid (mp, 117-119jC). 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 aluminum backing, 7:3
`hexane/ethyl acetate, R f = 0.66. 1H NMR (CDCl3) y 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 Hz), 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). [a]24
`D (c 0.106, DMF) = -11.5; FAB+ m/z
`520.2135 [MH+].
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`Rodent biodistribution
`The xenograft-bearing mice (17-20 g) were injected via the tail vein
`with 3.70 MBq (100 ACi, 286 pmol, 350 Ci/mmol) of [18F]DCFBC in
`200 AL 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 after injection (n = 4 per time point). Tissue radio-
`pharmaceutical uptake values were calculated as percent injected dose
`per gram (% ID/g) compared with a 1:10 diluted standard dose. The
`urinary bladder was emptied and water washed and then dried before
`weighing and counting.
`
`Small-animal PET
`A severe combined immunodeficient mouse bearing s.c. 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). The mouse was
`injected i.v. with 7.4 MBq (200 ACi, 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 2 h). The dwell time at each position was
`5 min such that a given bed position (or mouse organ) was revisited
`every 10 min. An energy window of 250 to 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 were 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 and then fitting a monoexponential curve to the last three
`time points to extrapolate effective clearance rate beyond the last
`measured time point. The sum of the numerical (measured data) and
`the analytic 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.
`DCFBC is a small molecule whose concentration in blood is not
`readily related to that in marrow. The red marrow was not directly
`sampled. The absorbed dose to red marrow is therefore the cross-fire
`dose from all activity containing sources in the body; this includes
`activity in the trabecular bone, which was directly measured, and
`blood (assigned to rest of body term). 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. Figure 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 Na-Fmoc-S-tert-butyl-L-
`
`Imaging, Diagnosis, Prognosis
`
`(CD3OD) y 4.45 (t, J = 4.5 Hz, 1H), 4.21 (dd, J = 5, 8.5 Hz, 1H), 2.82
`(d, J = 4.5 Hz, 2H), 2.31 (m, 2H), 2.05 (m, 1H), 1.80 (m, 1H); [a]24
`D
`+13.3 (c 0.0247, MeOH), Lit. (13) [a]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.
`(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 60jC for 10 min and then acidified with TFA. Purification
`by reverse-phase HPLC (C-18 Econosil 10  250 mm preparative
`column; 32:68:0.1 CH3CN/H2O/TFA) gave pure product. 1H NMR
`(CD3OD) y 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].
`
`Radiochemistry
`N- [N -[(S )-1,3-dicarboxypropyl ]carbamoyl ]-4 -[18F ]fluorobenzyl-L-
`cysteine. Typically, starting with 12.95 GBq (350 mCi) of [18F]KF, a
`solution containing 91% 4-[18F]FBB (14) was produced in 45 min
`[total reactivity, 3.4 GBq (93 mCi)] and placed in a 10 mL screw-cap
`borosilicate glass vial. To this solution (1.5 mL), still containing the
`brominating agent triphenylphosphine dibromide in methylene chlo-
`ride/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 AL methanol had been added
`previously. The liquid was concentrated under a stream of nitrogen at
`room temperature to f200 AL. 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-mercaptoethyl]ureido]-pentanedioic acid
`(6; 2-4 mg/40 AL). To this was added 200 AL of methanol previously
`saturated with ammonia gas. The vial was sealed and heated at 65jC for
`10 min and then cooled for 2 min. To the reaction was added 600 AL
`water, 80 AL of TFA (a 5 AL sample was spotted on pH paper to confirm
`acidity), and 600 AL of HPLC mobile phase. The resulting product was
`then purified by reverse-phase semipreparative radio-HPLC using a
`mobile phase consisting of 35:65:0.1% acetonitrile/water/TFA 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 PBS (pH 7.4), and filtered through a
`0.22 Am syringe filter into an evacuated sterile vial.
`In vitro inhibition assay. Taking advantage of the N-acetylaspartyl-
`glutamate (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
`(also known as PSMA), 4 Amol/L NAAG as a substrate, and a trace
`amount of [3H]NAAG. Inhibitors at concentrations of 0.1, 1, 10, and
`100 nmol/L were tested. Product was separated using ion exchange
`chromatography (AG-50W-X8 analytic-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, Cleveland, OH) and
`maintained as previously described (16). All cells were grown to 80% to
`90% confluency before trypsinization and formulation in HBSS (Sigma-
`Aldrich) for implantation into mice.
`in full compliance with
`All animal studies were carried out
`institutional guidelines related to the conduct of animal experiments.
`Male severe combined immunodeficient mice (Charles River Labora-
`tories) were implanted s.c. with 1  106 to 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 to 5 mm in diameter.
`
`Clin Cancer Res 2008;14(10) May 15, 2008
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`[18F]DCFBC Synthesis and In vivo Evaluation
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`Fig. 1. a, 4-methoxybenzyl chloride, Cs2CO3, DMF. b, 20% piperidine, DMF. c, triphosgene, triethylamine followed by 3. d, 1, TFA, anisole, Hg(OAc)2; 2, H2S.
`e, ammonia-saturated methanol, 4-fluorobenzyl iodide.
`
`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 60jC 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 F 6% (n = 8). In a typical experiment, the
`non – decay-corrected yield of the total synthesis from [18F]KF
`was 3.5% in 123 min (decay-corrected yield was 7.6%).
`The specific radioactivities ranged from 13 to 133 GBq/Amol
`(350-3,600 Ci/mmol) with an average of 52 GBq/Amol
`(1,492 Ci/mmol; n = 6). The range of specific radioactivities,
`
`Table 1. Tissue distribution of [18F]DCFBC
`
`Tissue
`
`Blood
`Heart
`Lung
`Liver
`Stomach
`Spleen
`Fat
`Kidney
`Bone
`Muscle
`Small intestine
`Large intestine
`Bladder (empty)
`PC-3 FLU tumor
`PC-3 PIP tumor
`
`*n = 3.
`
`5 min
`
`11.3 F 6.7
`4.4 F 0.9
`7.0 F 0.5
`6.0 F 1.2
`2.9 F 0.3
`8.8 F 1.3
`1.8 F 0.4
`63.4 F 8.2
`2.9 F 0.4
`2.2 F 1.0
`4.3 F 0.4
`2.8 F 0.4
`55 F 30
`3.5 F 0.5
`8.2 F 0.8
`
`15 min
`
`4.1 F 2.5
`2.0 F 0.6
`3.2 F 0.9
`4.1 F 1.4
`1.4 F 0.3
`4.3 F 1.4
`1.6 F 1.0
`63 F 20
`1.6 F 0.3
`1.0 F 0.4
`2.3 F 1.3
`1.5 F 0.7
`16 F 15
`1.7 F 0.4
`6.0 F 2.6
`
`%ID/g F SD (n = 4)
`
`30 min
`
`2.3 F 1.3
`1.2 F 0.2
`1.8 F 0.3
`4.2 F 0.5
`0.8 F 0.1
`1.9 F 0.9
`0.7 F 0.5
`51.3 F 7.5
`1.7 F 0.1
`0.5 F 0.1
`1.2 F 0.2
`0.8 F 0.1
`15 F 15
`1.0 F 0.1
`6.2 F 1.0
`
`60 min
`
`1.8 F 1.4
`0.8 F 0.3
`1.1 F 0.4
`5.1 F 0.8
`0.5 F 0.2
`1.6 F 0.9
`1.0 F 0.7
`41.6 F 7.2
`1.7 F 0.8
`0.6 F 0.5
`0.7 F 0.2
`0.6 F 0.2
`14.5 F 7.3
`0.8 F 0.2
`8.2 F 2.5
`
`120 min*
`
`0.4 F 0.2
`0.3 F 0.2
`0.4 F 0.1
`2.1 F 1.4
`1.1 F 1.9
`0.4 F 0.2
`0.3 F 0.1
`13 F 10
`2.5 F 3.6
`0.2 F 0.4
`0.2 F 0.1
`0.3 F 0.1
`2.6 F 1.0
`0.2 F 0.0
`4.7 F 0.9
`
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`
`3039
`
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`
`Imaging, Diagnosis, Prognosis
`
`Table 2. PC-3 PIP tumor to tissue ratios
`
`Tissue
`
`5 min 15 min 30 min 60 min 120 min*
`
`0.7
`1.9
`1.2
`1.4
`2.8
`0.9
`4.7
`0.1
`2.8
`3.7
`1.9
`2.9
`0.15
`2.4
`
`Blood
`Heart
`Lung
`Liver
`Stomach
`Spleen
`Fat
`Kidney
`Bone
`Muscle
`Small intestine
`Large intestine
`Bladder (empty)
`FLU tumor
`
`*n = 3.
`
`1.5
`3.0
`1.9
`1.5
`4.5
`1.4
`3.9
`0.1
`3.8
`6.0
`2.6
`4.1
`0.4
`3.5
`
`2.7
`5.0
`3.5
`1.5
`7.6
`3.2
`9.2
`0.1
`3.6
`14
`5.1
`7.9
`0.4
`6.4
`
`4.5
`10
`7.4
`1.6
`18
`5.2
`7.8
`0.2
`4.7
`14
`12
`14
`0.6
`11
`
`13
`17
`30
`2.2
`4.3
`11
`18
`0.4
`1.9
`20
`31
`17
`1.8
`27
`
`although wide, represents data from all syntheses done,
`including the first. However, even with this unoptimized
`manual
`technique, we have been able to achieve specific
`radioactivities that should be sufficient for human studies.
`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 to 100 nmol/L against a fixed amount of NAAG
`(4 Amol/L) 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]glu-
`tamate, was measured by scintillation counting and plotted
`against the logarithmic concentration of DCFBC. Linear regres-
`sion of the resulting data was solved for 50% [3H]glutamate
`(50% inhibition) and resulted in an IC50 value of 13.9 nmol/L
`
`for DCFBC. That result is in keeping with other compounds of
`this class (7, 8, 13).
`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 5-min postinjection time point. By
`60 min after injection, 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 partially due to specific binding (18, 19) but are
`likely dominated by renal clearance because washout was much
`faster from kidney than PC-3 PIP tumor. Urinary bladder
`uptake represents excretion at all time points (i.e., there was no
`specific binding to bladder wall), whereas tumor uptake shows
`a high degree of specificity represented by the PIP to 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.
`Figure 2 shows the PET scans at specific time intervals.
`Specific uptake in PSMA+ PIP tumor is clearly seen as early as
`the 20- to 30-min image. Clearance from nontarget tissues is
`evident at later time points. By 2 h, 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 (Fig. 3).
`Although the blood curve seems to decrease from 30 to 60 min,
`those from liver and tumor seem to rise. That is an unexpected
`and likely artifactual result due to the large SE in determining
`the %ID/g in those tissues. Little nonspecific tissue radioactivity
`uptake is evident. Renal excretion dominates with this
`hydrophilic compound.
`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)].
`
`Fig. 2. GE eXplore Vista dynamic PET images. Animals were injected with 7.4 MBq (200 ACi, 572 pmol, 350 Ci/mmol) of [18F]DCFBC before imaging. Summation
`images from the indicated time frames are shown. The PSMA+ PC-3 PIP tumor is under the left shoulder, whereas the PSMA- PC-3 tumor is under the right shoulder. Note
`accumulation of [18F]DCFBC only within the target-containing tumor. Radioactivity intensifies within the tumor and begins to wash out of the kidneys on the time scale
`shown.
`
`Clin Cancer Res 2008;14(10) May 15, 2008
`
`3040
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`
`[18F]DCFBC Synthesis and In vivo Evaluation
`
`membrane antigen (26). PSMA is up-regulated in prostate
`cancer, particularly in advanced, hormone-independent, and
`metastatic disease (27, 28). It is also expressed in the neo-
`vasculature 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). PSMA 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 (29 – 32).
`There is an emerging literature on the development of inhi-
`bitors for PSMA, which is expected to increase with the recent
`availability of a high-resolution crystal structure of the enzyme
`(13, 33 – 35). We and others have synthesized radiopharma-
`ceuticals and optical agents for PSMA detection (7 – 9, 36, 37),
`with [18F]DCFBC representing the first agent designed speci-
`fically for clinical PET imaging of prostate cancer.
`[18F]DCFBC localized selectively to the PSMA+ prostate
`tumor, PC-3 PIP (Fig. 2; Tables 1 and 2), achieving a target/
`background (muscle) ratio of 20:1 at 120 min after injection.
`The time-activity curves indicate that [18F]DCFBC has achieved
`equilibrium by 120 min and has begun to decrease in concen-
`tration 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 ratio in s.c.
`LNCaP tumors, and a similar uptake ratio shown in the clinic
`provided a positive predictive value for the presence of prostate
`cancer at >90% (38). [18F]DCFBC did not show 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-h PIP/
`bone value was 4.7, suggesting that metastatic lesions, which
`are known to express PSMA, will be clearly delineated (11, 39).
`As seen for our initial PET radioligand, [11C]DCMC (7), there
`is significant uptake within kidney that is due, at least in part, to
`specific binding to proximal renal tubules. We have shown this
`
`Table 3. Radiation absorbed doses
`
`Total absorbed doses
`(mGy/MBq)
`
`Brain
`Lower large intestinal wall
`Small intestine
`Stomach wall
`Upper large intestinal wall
`Heart wall
`Kidneys
`Liver
`Lungs
`Muscle
`Pancreas
`Red marrow
`Spleen
`Urinary bladder wall
`Total body
`
`4.97E-04
`1.01E-03
`1.45E-03
`1.57E-03
`1.53E-03
`2.25E-03
`4.87E-02
`9.34E-03
`2.58E-03
`1.37E-03
`3.03E-03
`3.12E-03
`4.25E-03
`8.99E-04
`1.79E-03
`
`NOTE: Estimate reflects dose contribution from other organs.
`Absorbed dose values in mGy/MBq per organ expected in average
`human.
`
`Fig. 3. Time-activity curves of [18F]DCFBC biodistribution in selected tissues
`(ex vivo analysis). Animals were injected with 3.70 MBq (100 ACi, 286 pmol,
`350 Ci/mmol) of [18F]DCFBC before sacrifice and harvest of the indicated tissues.
`There is a peak of radioactivity within tumor and muscle at 60 min after injection
`before washout (n = 3).
`
`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.
`
`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. Although it is true that nomograms, composed of
`clinical variables 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 in