articles
`
`Prostate-Specific Membrane Antigen Targeted Imaging
`and Therapy of Prostate Cancer Using a PSMA Inhibitor
`as a Homing Ligand
`
`Sumith A. Kularatne,† Kevin Wang,‡ Hari-Krishna R. Santhapuram,‡ and
`Philip S. Low*,†
`
`Department of Chemistry, Purdue UniVersity, 560 OVal DriVe, West Lafayette, Indiana
`47907, and Endocyte, Inc., 3000 Kent AVenue, West Lafayette, Indiana 47906
`
`Received February 25, 2009; Revised Manuscript Received April 9, 2009; Accepted April 11, 2009
`
`Abstract: Prostate cancer (PCa) is a major cause of mortality and morbidity in Western society
`today. Current methods for detecting PCa are limited,
`leaving most early malignancies
`undiagnosed and sites of metastasis in advanced disease undetected. Major deficiencies also
`exist in the treatment of PCa, especially metastatic disease. In an effort to improve both detection
`and therapy of PCa, we have developed a PSMA-targeted ligand that delivers attached imaging
`and therapeutic agents selectively to PCa cells without targeting normal cells. The PSMA-targeted
`radioimaging agent (DUPA-99mTc) was found to bind PSMA-positive human PCa cells (LNCaP
`cell line) with nanomolar affinity (KD ) 14 nM). Imaging and biodistribution studies revealed that
`DUPA-99mTc localizes primarily to LNCaP cell tumor xenografts in nu/nu mice (% injected dose/
`gram ) 11.3 at 4 h postinjection; tumor-to-muscle ratio ) 75:1). Two PSMA-targeted optical
`imaging agents (DUPA-FITC and DUPA-rhodamine B) were also shown to efficiently label
`PCa cells and to internalize and traffic to intracellular endosomes. A PSMA-targeted chemo-
`therapeutic agent (DUPA-TubH) was demonstrated to kill PSMA-positive LNCaP cells in culture
`(IC50 ) 3 nM) and to eliminate established tumor xenografts in nu/nu mice with no detectable
`weight loss. Blockade of tumor targeting upon administration of excess PSMA inhibitor (PMPA)
`and the absence of targeting to PSMA-negative tumors confirmed the specificity of each of the
`above targeted reagents for PSMA. Tandem use of the imaging and therapeutic agents targeted
`to the same receptor could allow detection, staging, monitoring, and treatment of PCa with
`improved accuracy and efficacy.
`
`Keywords: Prostate-specific membrane antigen; PSMA-targeted imaging and therapy; radio-
`imaging and optical imaging of prostate cancer; tubulysin prodrug; diagnosis of prostate cancer;
`chemotherapy for prostate cancer
`
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`
`Introduction
`Prostate cancer (PCa) is the most common male malig-
`nancy in western society, amounting to ∼230,000 new cases/
`year in the US.1 More males die from PCa (>30,000/year)
`than any other malignancy except lung cancer,1 and the
`
`* Corresponding author. Mailing address: Department of Chem-
`istry, Purdue University, 560 Oval Drive, West Lafayette, IN
`47907. Phone: 765-494-5273. Fax: 765-494-5272. E-mail:
`plow@purdue.edu.
`† Purdue University.
`‡ Endocyte, Inc.
`
`780 MOLECULAR PHARMACEUTICS VOL. 6, NO. 3, 780–789
`
`cumulative cost of treating PCa patients has been estimated
`at $8-10 billion/year in the US.2 Advanced stages of PCa
`can also significantly impact quality of life due to bone
`disintegration, pain, obstruction of urination, and erectile
`dysfunction among other disorders.3
`Although neoplastic transformation begins in the prostate
`gland, malignant cells can eventually metastasize to other
`parts of the body, including bones, rectum, and bladder.
`Because metastatic PCa is difficult to treat, early detection
`
`(1) Jemal, A.; Siegel, R.; Ward, E. Cancer statistics 2008. CasCancer
`J. Clin. 2008, 58, 71–96.
`
`10.1021/mp900069d CCC: $40.75  2009 American Chemical Society
`Published on Web 04/12/2009
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`

`

`PSMA-Targeted Diagnosis and Therapy of Prostate Cancer
`
`articles
`
`constitutes the most effective strategy for minimizing disease-
`related morbidity and mortality. Early diagnosis of PCa is
`most commonly achieved by digital rectal exam, blood test
`for prostate specific antigen (PSA), or a prostate biopsy.4
`However, only more advanced stages of disease can be
`detected by a digital rectal exam5 and prostate biopsies can
`be expensive and painful.6 Further, the accuracy of the PSA
`test has been criticized7 due to its elevation during benign
`prostatic hypertrophy (BPH) or prostatitis and due to its
`decline during treatment for BPH or baldness.8 Although
`transrectal ultrasound together with magnetic resonance
`imaging (MRI) and computerized tomography (CT) can
`effectively reveal the extent of prostate enlargement and
`growth asymmetry, these exams are too expensive for routine
`screening and may not distinguish malignant disease from
`BPH.9 Clearly, better methods for assessing onset and spread
`of PCa could greatly reduce the frequency of advanced stage
`disease.
`
`Treatment for PCa most commonly involves surgery,
`radiation therapy, hormone administration, and/or chemo-
`therapy. Unfortunately, none of these therapies is highly
`effective against metastatic disease, and each has sufficient
`disadvantages that patients often decline their use. While
`localized PCa can be treated by removal of malignant
`tissue,10 radical prostatectomy may result in loss of urinary
`control and impotence.11 Radiation therapy can also cause
`impotence, rectal bleeding,3 and increased risk of colon and
`
`(2) Wilson, L. S.; Tesoro, R.; Elkin, E. P. Cumulative cost pattern
`comparison of prostate cancer treatments. Cancer 2007, 109, 518–
`527.
`(3) Katz, A. Quality of life for men with prostate cancer. Cancer Nurs.
`2007, 30, 302–308.
`(4) Zeller, J. L. Grading of prostate cancer. JAMA, J. Am. Med. Assoc.
`2007, 298, 1596.
`(5) Chodak, M. D.; Keller, P.; Schoenberg, H. W. Assessment of
`screening for prostate cancer using the digital rectal examination.
`J. Urol. 1989, 141, 1136–1138.
`(6) Essink-Bot, M.; de Koning, H. J.; Nijs, H. G. J.; Kirkels, W. J.;
`van der Mass, P. J.; Schroder, F. H. Short
`term effects of
`population-based screening for prostate cancer on health-related
`quality of life. J. Natl. Cancer Inst. 1998, 90, 925–931.
`(7) Linn, M. M.; Ball, R. A.; Maradiegue, A. Prostate-specific antigen
`screening: Friend or foe? Urol. Nurs. 2007, 27, 481–489.
`(8) D’Amico, A. V.; Roehrborn, C. G. Effect of 1 mg/day finasteride
`on concentration of serum prostate specific antigen in men with
`androgenic alopecia: a randomized controlled trial. Lancet Oncol.
`2007, 8, 21–25.
`(9) Holves, A. M.; Heesakkers, R. A. M.; Adang, E. M. The diagnostic
`accuracy of CT and MRI in the pelvis of lymph nodes in patients
`with prostate cancer: a meta-analysis. Clin. Radiol. 2008, 63, 387–
`95.
`(10) Bill-Axelson, A.; Andersson, S.-O.; Bratell, S. Radical prostate-
`ctomy versus watchful waiting in early prostate cancer. N. Engl.
`J. Med. 2005, 352, 1977–1984.
`(11) Weber, B. A.; Robert, B. L.; Chumbler, N. R.; Mills, T. L.;
`Algood, C. B. Urinary, sexual and bowel dysfunction and bother
`after radical prostatectomy. Urol. Nurs. 2007, 27, 527–533.
`
`bladder cancer,12 and treatment of invasive or metastatic PCa
`is often limited to palliative hormonal
`therapy and/or
`chemotherapy. While hormonal treatment induces remission
`of hormonally responsive cancer, the longevity of tumor
`remission is limited and it is not without significant toxicity,
`including liver damage, cardiovascular disease, weight gain,
`and osteoporosis.13 And although chemotherapy (e.g., mi-
`toxantrone) may also extend lifespan,14 side effects of such
`antimitotic drugs often outweigh their benefits. Therefore,
`safer and more potent methods of treating PCa are widely
`needed.
`In an effort to improve both detection and treatment of
`PCa, we initiated a search for low molecular weight ligands
`that would selectively target attached drugs to PCa cells
`without promoting their uptake by healthy cells. Prostate-
`specific membrane antigen (PSMA, folate hydrolase I,
`glutamate carboxypeptidase II), a plasma membrane-associ-
`ated protein,15 is overexpressed on the vast majority of PCa.16
`While the physiological function of PSMA remains contro-
`versial, its expression is largely limited to PCa cells,16,17
`where malignant transformation leads not only to its up-
`regulation but also to its translocation from internal organelles
`to the cell surface. For unknown reasons, PSMA is also
`expressed in the neovasculature of most other solid tumors
`(but not in the vasculature of healthy tissues),16,18 and in
`the kidneys, albeit at significantly lower levels in human
`kidneys16,17 than murine kidneys.19 For drug targeting
`applications, perhaps the most important characteristic of
`PSMA is that it undergoes internalization through clathrin-
`coated pits and rapidly recycles to the cell surface for
`
`(12) Brenner, D. J.; Curtis, R. E.; Ron, E. Second malignancies in
`prostate carcinoma patients after radiotherapy compared with
`surgery. Cancer 2000, 88, 398–406.
`(13) Kumar, R. J.; Bargawi, A. Crawford ED. Adverse events
`associated with hormonal therapy for prostate cancer. ReV. Urol.
`2005, 7, 37–43.
`(14) Hsiao, C.; Li, T.-K.; Chan, Y.-L. WRC-213, an L-methionine-
`conjugated mitoxantrone derivative, displays anticancer activity
`with reduced cardiotoxicity and drug resistance: identification of
`topoisomerase II inhibition and apoptotic machinery. Biochem.
`Pharmacol. 2008, 75, 847–856.
`(15) Gang, M. C.; Chang, S. S.; Sadelain, M.; Bander, N. H.; Heston,
`W. D. Prostate specific membrane antigen (PSMA)-specific
`monoclonal antibodies in the treatment of prostate and other
`cancers. Cancer Metastasis ReV. 1999, 18, 483–490.
`(16) Ghosh, A.; Heston, W. D. Tumor target prostate specific mem-
`brane antigen (PSMA) and its regulation in prostate cancer. J. Cell
`Biochem. 2004, 91, 528–539.
`(17) Silver, D. A.; Pellicer, I.; Fair, W. R.; Heston, W. D.; Cordon-
`Cardo, C. Prostate-specific membrane antigen expression in
`normal and malignant human tissues. Clin. Cancer Res. 1997, 3,
`81–85.
`(18) Chang, S. S.; O’Keefe, D. S.; Bacich, D. J.; Reuter, V. E.; Heston,
`W. D.; Gaudin, P. B. Prostate-specific membrane antigen is
`produced in tumor-associated neovasculature. Clin. Cancer Res.
`1999, 5, 2674–2681.
`(19) Slusher, B. S.; Tsai, G.; Yoo, G.; Coyle, J. T. Immunocytochemical
`localization of the N-acetyl-aspartyl-glutamate (NAAG) hydrolyz-
`ing enzyme N-acetylated R-linked acidic dipeptidase (NAALA-
`Dase). J. Comp. Neurol. 1992, 315, 217–229.
`
`VOL. 6, NO. 3 MOLECULAR PHARMACEUTICS 781
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`

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`articles
`
`Kularatne et al.
`
`additional rounds of internalization.20 Collectively, these
`unique features render PSMA an excellent candidate for use
`in tumor-targeted drug delivery.
`For the above reasons, a variety of efforts have been made
`to develop PSMA-targeted imaging agents for use in the
`diagnosis and monitoring of PCa.21-27 Indeed, a variety of
`low molecular weight
`inhibitors of PSMA have been
`radiolabeled and used to image human PCa xenografts
`(LNCaP and PC-3 cell lines) in athymic nude mice.23,28-30
`In this report, we describe the design and use of a PSMA-
`specific ligand for the selective delivery of both imaging and
`therapeutic agents to a human PCa xenograft (LNCaP cells)
`in nu/nu mice. We also demonstrate that the PSMA-specific
`ligand can deliver sufficient tubulysin, a microtubule inhibi-
`tor, to established solid LNCaP tumors in athymic mice to
`induce their long-term remission without causing measurable
`toxicity to healthy tissues.
`
`Experimental Section
`Materials. Sodium pertechnetate was purchased from
`Cardinal Health (Indianapolis, IN). [3H]-Thymidine was
`
`(20) Liu, H.; Rajasekaran, A. K.; Moy, P. Constitutive and antibody-
`induced internalization of prostate-specific membrane antigen.
`Cancer Res. 1998, 58, 4055–4060.
`(21) Sodee, D. B.; Ellis, R. J.; Samuels, M. A. Prostate cancer and
`prostate bed SPECT imaging with ProstaScint: Semiquantitative
`correlation with prostatic biopsy results. Prostate 1998, 37, 140–
`148.
`(22) Milowsky, M. I.; Nanus, D. M.; Kostakoglu, L. Vascular targeted
`therapy with anti-prostate-specific membrane antigen monoclonal
`antibody J591 in advanced solid tumors. J. Clin. Oncol. 2007,
`25, 540–547.
`(23) Zhou, J.; Neale, J. H.; Pomper, M. G.; Kozikowski, A. P. NAAG
`peptidase inhibitors and their potential for diagnosis and therapy.
`Nat. ReV. 2005, 4, 1015–1026.
`(24) Misra, P.; Valerie, H.; Pannier, N.; Maison, W.; Frangioni, J. V.
`Production of multimeric prostate-specific membrane antigen
`small-molecule radiotracers using a solid-phase 99mTc preloading
`strategy. J. Nucl. Med. 2007, 48, 1379–1389.
`(25) Tang, H.; Brown, M.; Ye, Y. Prostate targeting ligands based on
`N-acetylated a´-linked acidic dipeptidase. Biochem. Biophys. Res.
`Commun. 2003, 307, 8–14.
`(26) Humblet, V.; Lapidus, R.; Williams, L. High-affinity near-infrared
`fluorescent small-molecule contrast agents for in vivo imaging
`of prostate-specific membrane antigen. Mol. Imaging 2005, 4,
`448–462.
`(27) Liu, T.; Wu, L.; Kazak, M.; Berkman, C. E. Cell-surface labeling
`and internalization by a fluorescent inhibitor of prostate-specific
`membrane antigen. Prostate 2008, 68, 955–964.
`(28) Mease, R. C.; Dusich, C. L.; Foss, C. A. N-[N-[(S)-1,3-
`dicarboxypropyl]carbamoyl]-4-[18F]fluorobenzyl-Lcysteine, [18F]D-
`CFBC: a new imaging probe for prostate cancer. Clin. Cancer
`Res. 2008, 14, 3036–3043.
`(29) Banerjee, S. R.; Foss, A. C.; Castanares, M. Synthesis and
`evaluation of technetium-99m- and rhenium-labeled inhibitors of
`the prostate-specific membrane antigen (PSMA). J. Med. Chem.
`2008, 51, 4504–4517.
`(30) Chen, Y.; Foss, C. A.; Byun, Y. Radiohalogenated prostate-specific
`membrane antigen (PSMA)-based ureas as imaging agents for
`prostate cancer. J. Med. Chem. 2008, 51, 7933–7943.
`
`782 MOLECULAR PHARMACEUTICS VOL. 6, NO. 3
`
`obtained from Moravek Biochemicals (Brea, CA), and
`2-(phosphonomethyl)-pentanedioic acid (PMPA) was from
`Axxora Platform (San Diego, CA). Tubulysin B was
`provided by Endocyte Inc. (W. Lafayette, IN). HC Matrigel
`was obtained from BD Bioscience (San Jose, CA). All other
`chemicals were purchased from major suppliers.
`Synthesis of DUPA Conjugates. DUPA-99mTc was
`synthesized as described in the companion paper (DOI
`10.1021/mp9000712) to this report.31 The synthesis of
`DUPA-TubH will be described in detail elsewhere (manu-
`script in preparation). DUPA-FITC was synthesized by solid
`phase methodology as follows (see Scheme 1). Universal
`NovaTag resin (50 mg, 0.53 mM) was swollenwith dichlo-
`romethane (DCM) (3 mL) followed by dimethylformamide
`(DMF, 3 mL). A solution of 20% piperidine in DMF (3 ×
`3 mL) was added to the resin, and argon was bubbled for 5
`min. The resin was washed with DMF (3 × 3 mL) and
`isopropyl alcohol (i-PrOH, 3 × 3 mL). After swelling the
`resin in DMF, a solution of DUPA(OtBu)-OH (1.5 equiv),
`HATU (2.5 equiv) and DIPEA (4.0 equiv) in DMF was
`added. Argon was bubbled for 2 h, and resin was washed
`with DMF (3 × 3 mL) and i-PrOH (3 × 3 mL). After
`swelling the resin in DCM, a solution of 1 M HOBt in DCM/
`trifluoroethane (TFE) (1:1) (2 × 3 mL) was added. Argon
`was bubbled for 1 h, the solvent was removed and resin was
`washed with DMF (3 × 3 mL) and i-PrOH (3 × 3 mL).
`After swelling the resin in DMF, a solution of Fmoc-Phe-
`OH (2.5 equiv), HATU (2.5 equiv) and DIPEA (4.0 equiv)
`in DMF was added. Argon was bubbled for 2 h, and resin
`was washed with DMF (3 × 3 mL) and i-PrOH (3 × 3 mL).
`The above sequence was repeated for 2 more coupling steps
`for addition of 8-aminooctanoic acid and fluorescein isothio-
`cyanate or rhodamine B isothiocyanate. Final compound was
`cleaved from the resin using a trifluoroacetic acid (TFA):
`H2O:triisopropylsilane:cocktail (95:2.5:2.5) and concentrated
`under vacuum. The concentrated product was precipitated
`in diethyl ether and dried under vacuum. The crude product
`was purified using preparative RP-HPLC [λ ) 488 nm;
`solvent gradient: 1% B to 80% B in 25 min, 80% B wash
`30 min run; A ) 10 mM NH4OAc, pH ) 7; B ) acetonitrile
`(ACN)]. ACN was removed under vacuum, and pure
`fractions were freeze-dried to yield DUPA-FITC as a
`brownish-orange solid. RP-HPLC: tR ) 8.0 min (A ) 10
`mM NH4OAc, pH ) 7.0; B ) ACN, solvent gradient: 1%
`
`(31) Kularatne, S. A.; Zhou, Z.; Yang, J.; Post, C. B.; Low, P. S.
`Design, synthesis, and preclinical evaluation of prostate-specific
`membrane antigen targeted 99mTc-radioimaging agents Mol. Phar-
`maceutics. DOI 10.1021/mp9000712.
`(32) Leamon, C. P.; Parker, M. A.; Vlahoc, I. R. Synthesis and
`biological evaluation of EC20: a new folate-derived 99mTc-based
`radiopharmaceutical. Bioconjugate Chem. 2002, 13, 1200–1210.
`(33) Leamon, C. P.; Reddy, J. A.; Wetzel, M. Folate targeting enables
`durable and specific antitumor responses from a therapeutically
`null tubulysin B analogue. Cancer Res. 2008, 68, 9839–9844.
`(34) He, W.; Kularatne, S. A.; Kelli, K. R. Quantitation of circulating
`tumor cells in blood samples from ovarian and prostate cancer
`patients using tumor-specific fluorescent ligands. Int. J. Cancer
`2008, 123, 1968–1973.
`
`

`

`PSMA-Targeted Diagnosis and Therapy of Prostate Cancer
`
`articles
`
`Scheme 1a
`
`a Reagents and conditions: (a) (i) 20% piperidine/DMF, rt, 10 min; (ii) DUPA(OtBu)-OH, HATU, DIPEA, 2 h; (b) 1 M HOBt in DCM/TFE (1:1), 1 h; (c)
`(i) 20% piperidine/DMF, rt, 10 min; (ii) Fmoc-Phe-OH, HATU, DIPEA, 2 h; (d) (i) 20% piperidine/DMF, rt, 10 min; (ii) Fmoc-8-aminooctanoic (EAO)
`acid, HATU, DIPEA, 2 h; (e) (i) 20% piperidine/DMF, rt, 10 min; (ii) fluorescein isothiocyanate (FITC) or rhodamine B isothiocyanate, DIPEA/DMF; (f)
`TFA/H2O/TIPS (95:2.5:2.5), 30 min.
`
`B to 50% B in 10 min, 80% B wash 15 min run). 1H NMR
`(DMSO-d6/D2O): δ 0.98-1.27 (ms, 9H); 1.45 (b, 3H);
`1.68-1.85 (ms, 11H); 2.03 (m, 8H); 2.6-3.44 (ms, 12H);
`3.82 (b, 2H); 4.35 (m, 1H); 6.53 (d, J ) 8.1 Hz, 2H), 6.61
`(dd, J ) 5.3, 3.5 Hz, 2H); 6.64 (s, 2H); 7.05 (d, J ) 8.2 Hz,
`2H), 7.19 (m, 5H); 7.76 (d, J ) 8.0 Hz, 1H); 8.38 (s, 1H).
`HRMS (ESI) (m/z): (M + H)+ calcd for C51H59N7O15S,
`1040.3712, found, 1040.3702. UV/vis: λ max ) 491 nm.
`Characterization of DUPA-Rhodamine B. Purple solid,
`analytical HPLC: tR ) 8.4 min (A ) 10 mM NH4OAc, pH
`) 7.0; B ) CAN, solvent gradient: 1% B to 70% B in 10
`min, 80% B wash 15 min run); 1.08-1.53 (21H); 1.65 (b,
`4H); 1.84-2.20 (ms, 6H); 2.83 (m, 1H); 2.97 (b, 3H); 3.01
`(b, 3H); 3.36 (b, 9H); 3.90 (b, 2H); 4.62 (s, 1H); 6.52 (m,
`5H); 7.18 (m, 5H); 7.82 (m, 2H); 8.20 (m, 1H); 8.35 (s, 1H).
`LRMS (ESI) (m/z): (M)+ calcd for C59H76N9O13S, 1151.35,
`found, 1151.19. UV/vis: λ max ) 555 nm.
`Cell Culture and Animals. LNCaP, KB, and A549 cells
`were obtained from American Type Culture Collection. Cells
`were grown as a monolayer using 1640 RPMI medium
`containing 10% heat-inactivated fetal bovine serum, sodium
`pyruvate (100 mM) and 1% penicillin streptomycin in a 5%
`CO2:95% air-humidified atmosphere at 37 °C.
`Male nu/nu mice were purchased from NCI Charles River
`Laboratories and maintained on normal rodent diet during
`the study.
`Preparation of DUPA-99mTc Radioimaging Agent. A
`solution of sodium pertechnetate (1.0 mL, 15 mCi) was added
`to a vial containing a lyophilized mixture of DUPA-chelate
`
`conjugate (0.14 mg), sodium R-D-glucoheptonate dihydrate
`(80 mg), stannous chloride dihydrate (0.80 mg), and sufficient
`NaOH to achieve a pH of 7.2 upon rehydration with water.
`The vial was heated in a boiling water bath for 18 min and
`then cooled to rt before use.
`Binding Affinity and Specificity of DUPA-99mTc. LN-
`CaP cells (150,000 cells/well in 500 µL) were seeded into
`24-well Falcon plates and allowed cells to form monolayers
`over 48 h. Spent medium in each well was replaced with
`fresh medium (0.5 mL) containing increasing concentrations
`of DUPA-99mTc in the presence or absence of 100-fold
`excess (ex) PMPA. After incubating for 1 h at 37 °C, cells
`were rinsed with medium (2× 1.0 mL) and tris buffer (1×
`1.0 mL). After dissolving cells in 0.25 M NaOH(aq) (0.5 mL),
`cells were transferred into individual γ-counter tubes and
`radioactivity was counted using a γ-counter (Packard,
`Packard Instrument Company). KD was calculated by plotting
`bound radioactivity versus the concentration of radiotracer
`using GraphPad Prism 4.
`Flow Cytometry. LNCaP cells were seeded into a T75
`flask and allowed to form a monolayer over 48 h. After
`trypsin digestion, release cells were transferred into centrifuge
`tubes (1 × 106 cells/tube) and centrifuged. The medium was
`replaced with fresh medium containing DUPA-FITC (100
`nM) in the presence or absence of 100-fold excess PMPA
`and incubated for 1 h at 37 °C. After rinsing with fresh
`medium (2× 1.0 mL) and tris buffer (1× 1.0 mL), cells were
`resuspended in PBS (1.0 mL) and cell bound fluorescence
`was analyzed (500,000 cells/sample) using a flow cytometer
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`Kularatne et al.
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`(Cytomics F500, Beckman Coulter). Untreated LNCaP cells
`in PBS served as a negative control.
`Confocal Microscopy. LNCaP cells (100,000 cells/well
`in 1 mL) were seeded into poly-D-lysine microwell Petri
`dishes and allowed cells to form monolayers over 24 h. Spent
`medium was replaced with fresh medium containing
`DUPA-FITC (100 nM) or DUPA-rhodamine (100 nM) in
`the presence or absence of 100-fold excess PMPA and cells
`were incubated for 1 h at 37 °C. After rinsing with fresh
`medium (2× 1.0 mL) and PBS (1× 1.0 mL), confocal images
`were acquired using a confocal microscopy (FV 1000,
`Olympus).
`Tumor Models, Imaging, and Biodistribution Studies.
`Five-week-old male nu/nu mice were inoculated subcutane-
`ously with LNCaP cells (5.0 × 106/mouse in 50% HC
`Matrigel), KB cells, or A549 (1.0 × 106/mouse in RPMI
`medium) on their shoulders. Growth of the tumors was
`measured in two perpendicular directions every 2 days using
`a caliper (body weights were monitored on the same
`schedule), and the volumes of the tumors were calculated
`as 0.5 × L × W2 (L ) longest axis and W ) axis
`perpendicular to L in millimeters). Once tumors reached
`between 400 and 500 mm3 in volume, animals were treated
`with DUPA-99mTc (67 nmol, 150 µCi) in saline (100 µL).
`After 4 h, animals were sacrificed by CO2 asphyxiation.
`Images were acquired by a Kodak Imaging Station (In-Vivo
`FX, Eastman Kodak Company) in combination with CCD
`camera and Kodak molecular imaging software (version 4.0).
`Radioimages: illumination source ) radio isotope, acquisition
`time ) 3 min, f-stop ) 4, focal plane ) 5, FOV ) 160,
`binning ) 4. White light images: illumination source ) white
`light transillumination, acquisition time ) 0.05 s, f-stop )
`16, focal plane ) 5, FOV ) 160 with no binning.
`Following imaging, animals were dissected and selected
`tubes.
`tissues were collected to preweighed γ-counter
`Radioactivity of preweighed tissues and DUPA-99mTc (67
`nmol, 150 µCi) in saline (100 µL) was counted in a
`γ-counter. CPM values were decay corrected, and results
`were calculated as % ID/gram of wet tissue and tumor-to-
`tissue ratios.
`In Vitro Potency of DUPA-TubH. LNCaP cells were
`seeded into 24-well (50,000 cells/well in 500 µL) Falcon
`plates and allowed cells to form monolayers over 48 h. Spent
`medium was replaced with fresh medium (0.5 mL) containing
`increasing concentrations of DUPA-TubH in the presence
`or absence of 100-fold excess PMPA, and cells incubated
`for an additional 2 h at 37 °C. Cells were washed 3× with
`fresh medium and incubated in fresh medium (0.5 mL) for
`66 h at 37 °C. Spent medium in each well was replaced with
`fresh medium (0.5 mL) containing [3H]-thymidine (1 µCi/
`mL), and cells were incubated for 4 h at 37 °C to allow
`[3H]-thymidine incorporation. Cells were then rinsed with
`medium (3× 0.5 mL) and treated with 5% trichloroacetic
`acid (0.5 mL) for 10 min at rt. Cells were dissolved in 0.25
`M NaOH (0.5 mL), transferred into individual vials contain-
`ing Ecolume scintillation cocktail (3.0 mL), and counted in
`a scintillation counter (Packard, Packard Instrument Com-
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`pany). IC50 was calculated by plotting % 3H-thymidine
`incorporation versus log concentration of DUPA-TubH
`using GraphPad Prism 4.
`In Vivo Potency of DUPA-TubH. Healthy male nu/nu
`mice were administered with multiple doses of freshly
`prepared DUPA-TubH dissolved in saline (200 µL) via
`lateral tail injection on days zero, 2, 4, 6, 8, and 10. Body
`weights and clinical observations were monitored prior to
`dosing and daily thereafter from day zero to 12. Chronic
`maximum tolerance dose was determined by plotting %
`weight change versus days on therapy, and any animals with
`a body weight loss of 20% or more over two consecutive
`days were euthanized.
`Male nude mice bearing LNCaP xenograft tumors were
`then treated with 1.5 µmol/kg DUPA-TubH (for tumors of
`90-130 mm3) or 2.0 µmol/kg DUPA-TubH (for tumors of
`320-360 mm3) dissolved in 200 µL of saline via lateral tail
`vein injection. Treatments were conducted 3× per week for
`two weeks. Tumor volumes and body weights were measured
`on the same schedule. In vivo efficacy was evaluated by
`plotting tumor volume versus days on therapy.
`
`Results
`Design and Labeling of a PSMA-Targeted Radiotracer. In
`an effort to identify a high affinity targeting ligand for
`delivery of attached imaging and therapeutic agents to
`PSMA-expressing PCa cells, we conducted in silico docking
`studies on a series of PSMA inhibitors using a high resolution
`crystal structure of GCP-II in complex with the PSMA
`inhibitor termed GPI-18431 (PDB ID code 2C6C).35 Due to
`its favorable binding mode (in silico), ease of synthesis, high
`experimental affinity for purified PSMA (Ki ) 8 nM),36 and
`availability of a free carboxylic acid not required for PSMA
`binding, 2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid
`(DUPA, Figure 1a) was selected as a possible targeting
`ligand.
`Initial analysis of the tumor targeting specificity of DUPA-
`linked drugs required synthesis of a radiolabeled conjugate
`that would allow quantitation of its distribution in live
`animals. Because technetium (99mTc) is the major radioim-
`aging nuclide used clinically, a common chelator of 99mTc,
`diaminopropionic acid-Asp-Cys,32 was selected for attach-
`ment to DUPA. Thus, the PSMA crystal structure revealed
`a gradually narrowing access funnel 20 Å deep that extends
`from the protein surface to a binuclear zinc atom in the
`catalytic site.35 By modeling the peptide spacer to fit the
`contours of this funnel, we were able to generate a targeting
`moiety that did not compromise DUPA’s affinity for PSMA.
`Details and images of the fit of the peptide spacer to the 20
`
`(35) Mesters, J. R.; Barinka, C.; Li, W. Structure of glutamate
`carboxypeptidase II, a drug target in neuronal damage and prostate
`cancer. EMBO J. 2006, 25, 1375–1384.
`(36) Kozikowski, A. P.; Zhang, J.; Nan, F. Synthesis of urea-based
`inhibitors as active site probes of glutamate carboxypeptidase II:
`Efficacy as analgesic agents. J. Med. Chem. 2004, 47, 1729–1738.
`
`

`

`PSMA-Targeted Diagnosis and Therapy of Prostate Cancer
`
`articles
`
`Figure 1. Structures of (a) PSMA inhibitors, DUPA and PMPA, (b) PSMA-targeted radiotracer, DUPA-99mTc. (c) Binding
`affinity and specificity of DUPA-99mTc to PSMA-positive LNCaP cells in culture. Error bars represent SD (n ) 3).
`
`Å deep tunnel leading to the DUPA binding site are presented
`in a companion paper, DOI 10.1021/mp9000712.31
`Binding Affinity and Specificity of DUPA-99mTc. The
`affinity and specificity of the DUPA-99mTc radiotracer
`(Figure 1b) was first evaluated using LNCaP cells that
`express PSMA. The dissociation constant (KD) derived from
`these studies was calculated to be 14 nM (Figure 1c). This
`binding affinity compares well with the KI of DUPA for the
`purified enzyme (8 nM), suggesting that its conjugation to
`the chelating agent via the fitted peptide spacer does not
`extensively compromise its association with PSMA. The
`specificity of DUPA-99mTc for PSMA was next determined
`by coincubating LNCaP cells with the radioimaging agent
`in the presence of an excess (xs) of PMPA (blocked PSMA),
`a high affinity inhibitor of PSMA (KI ) 0.3 nM).37 PMPA
`competed quantitatively with DUPA-99mTc for binding to
`LNCaP cells (Figure 1c), demonstrating that PSMA consti-
`tutes the sole DUPA-99mTc binding site on this cell line.
`Binding and Internalization of Fluorescent Conjugates of
`DUPA. The ability of DUPA to deliver different cargos to
`PSMA on LNCaP cells was next evaluated by preparing
`DUPA conjugates of two fluorescent dyes and analyzing their
`binding and endocytosis by optical methods. LNCaP cells
`wereincubatedwithDUPA-FITC(Figure2a),DUPA-rhodamine
`B (Figure 2b), or DUPA-FITC in the presence of excess
`PMPA and analyzed by either flow cytometry or confocal
`fluorescence microscopy. DUPA-FITC was seen to ef-
`ficiently label LNCaP cells in a manner that could be
`inhibited by excess PMPA (Figure 2c), suggesting that the
`fluorescent conjugate’s binding was also PSMA-specific.
`More importantly, confocal microscopy revealed DUPA-FITC
`fluorescenceonlyatthecellsurface,whereasDUPA-rhodamine
`B fluorescence was seen throughout the LNCaP cell cyto-
`
`(37) Jackson, P. F.; Slusher, B. S. Design of NAALADase: A novel
`neuroprotective strategy. Curr. Med. Chem. 2001, 8, 949–957.
`
`plasm (Figure 2d). This apparent difference in intracellular
`distribution is informative, since FITC fluorescence is
`quenched at pHs < 6.2, while rhodamine B fluorescence is
`pH independent. Assuming both DUPA conjugates traffic
`through the same endosomes, these data suggest that DUPA
`conjugates are internalized and trafficked to acidic endosomes
`where FITC but not rhodamine B fluorescence is quenched.
`Whole Body Imaging of Solid Tumor Xenografts. To
`further establish the specificity of our DUPA conjugates for
`PCa cells, DUPA-99mTc was injected into nude mice bearing
`LNCaP tumors on their shoulders. The targeted 99mTc
`radiotracer accumulated mainly in the PSMA-positive LN-
`CaP tumor, with little or no radioactivity in other tissues
`except the kidneys (Figure 3a,c). Importantly, high levels of
`kidney uptake may be peculiar to the mouse, since immuno-
`cytochemical analyses and reported imaging studies of xenograft
`models suggest that PSMA expression is high in murine
`kidneys19,23,26,28-30 but low in human kidneys.16-18,22
`In vivo specificity of the PSMA-targeted imaging agent
`was further tested by prior administration of excess PMPA
`to block all PSMA sites
`(blocked PSMA) before
`DUPA-99mTc administration. Blocked LNCaP tumors dis-
`play no DUPA-99mTc uptake (Figure 3b), confirming the
`specificity of the DUPA conjugate for PSMA in vivo. To
`further document this specificity, the radiotracer was also
`administered to two PSMA-negative mouse xenograft models
`[A549 (a human lung cancer cell line) and KB (a human
`nasopharyngeal cancer cell line)], and again whole body
`images were taken. As anticipated, no radioactivity was
`observed in either KB or A549 tumors (data not shown).
`Even after shielding the kidneys to detect low levels of
`DUPA-99mTc, no radioactivity was found in PSMA-negative
`and blocked PSMA tumors (Figure 3d-f). These studies thus
`confirm that very little DUPA-99mTc binding occurs to sites
`unrelated to PSMA in vivo.
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`articles
`
`Kularatne et al.
`
`Figure 2. Structures of (a) DUPA-FITC and (b) DUPA-rhodamine B. Analysis of (c) binding of DUPA-FITC to
`LNCaP cells by flow cytometry and (d) binding and internalization of fluorescent DUPA conjugates to LNCaP cells by
`confocal microscopy in the presence and absence of excess PMPA. DIC ) differential interference contrast images.
`
`Figure 3. Overlay of whole-body radioimages on white light images of nu/nu mice bearing LNCaP, A549, or KB
`tumors 4 h after administration of DUPA-99mTc. In images (c-f), kidneys were shielded with lead pads. K ) kidney,
`red arrows ) solid tumor xenografts.
`
`Analysis of DUPA-99mTc Biodistribution in Vivo. After
`imaging the animals, biodistribution studies were also
`conducted to quantitate DUPA conjugate specificity for
`PSMA in vivo. For this purpose, animals were sacrificed 4 h
`after administration of DUPA-99mTc and tissues were
`analyzed for radioactivity in a γ-counter. The highest percent
`injected dose per gram of wet tissue (% ID/g) was observed
`in the tumor (11.2%) and kidneys (28.9%) (Figure 4). Uptake
`in both tissues was strongly competable upon preinjection
`of PMPA, confirming the prominent expression of PSMA
`in both tissues. All other normal
`tissues displayed
`DUPA-99mTc uptake levels of <1% ID/g, resulting in
`excellent tumor-to-normal tissue ratios (see Figure 1 in the
`Supporting Information) of 75:1 (tumor:muscle), 73:1 (tumor:
`heart), 29:1 (tumor:skin), 18:1 (tumor:liver) and 17:1 (tumor:
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`786 MOLECULAR PHARMACEUTICS VOL. 6, NO. 3
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`Figure 4. Biodistribution studies of DUPA-99mTc in nu/
`nu mice bearing LNCaP, A549, or KB tumors. Error
`bars represent SD (n ) 5 mice/group).
`
`spleen). The fact that the tumor:blood ratio was 33:1 also
`suggests that the DUPA-99mTc conjugate has low serum
`protein binding and clears rapidly from the blood.
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`PSMA-Targeted