68Ga-Complex Lipophilicity and the Targeting Property of a Urea-
`Based PSMA Inhibitor for PET Imaging
`Matthias Eder,*,† Martin Schä fer,† Ulrike Bauder-Wü st,† William-Edmund Hull,‡ Carmen Wä ngler,§
`Walter Mier,∥ Uwe Haberkorn,∥ and Michael Eisenhut†
`†Radiopharmaceutical Chemistry, ‡Molecular Structure Analysis, German Cancer Research Center, Heidelberg, Germany
`§Department of Nuclear Medicine, University Hospital Munich, Munich, Germany
`∥Department of Nuclear Medicine, University of Heidelberg, Heidelberg, Germany
`*S Supporting Information
`ABSTRACT: Urea-based inhibitors of the prostate-specific
`membrane antigen (PSMA) represent low-molecular-weight
`pepidomimetics showing the ability to image PSMA-expressing
`prostate tumors. The highly efficient, acyclic Ga(III) chelator
`N,N′-bis [2-hydroxy-5-(carboxyethyl)benzyl] ethylenediamine-
`N,N′- diacetic acid (HBED-CC) was introduced as a lipophilic
`side chain into the hydrophilic pharmacophore Glu-NH-CO-NH-
`Lys which was found favorable to interact with the PSMA“active
`binding site”. This report describes the syntheses, in vitro binding
`analyses, and biodistribution data of the radiogallium labeled
`PSMA inhibitor Glu-NH-CO-NH-Lys(Ahx)-HBED-CC in com-
`parison to the corresponding DOTA conjugate. The binding
`properties were analyzed using competitive cell binding and
`enzyme-based assays followed by internalization experiments. Compared to the DOTA-conjugate, the HBED-CC derivative
`showed reduced unspecific binding and considerable higher specific internalization in LNCaP cells. The
`68Ga complex of the
`HBED-CC ligand exhibited higher specificity for PSMA expressing tumor cells resulting in improved in vivo properties.68Ga
`labeled Glu-NH-CO-NH-Lys(Ahx)-HBED-CC showed fast blood and organ clearances, low liver accumulation, and high specific
`uptake in PSMA expressing organs and tumor. It could be demonstrated that the PET-imaging property of a urea-based PSMA
`inhibitor could significantly be improved with HBED-CC.
`■ INTRODUCTION
`It is still a challenge to select appropriate treatment options for
`disseminated prostate cancer because of the lack of sensitive
`imaging agents for diagnosis and therapy monitoring.
`1 Prostate-
`specific membrane antigen (PSMA) is expressed in nearly all
`prostate cancers with increased expression in poorly differ-
`entiated, metastatic, and hormone-refractory carcinomas.
`2 Its
`expression level is about 1000-fold higher compared to the
`physiologic levels found in other tissues such as kidney, small
`intestine, or brain.
`3 PSMA is primarily restricted to the prostate,
`it is abundantly expressed at all stages of disease, it is presented
`on the cell surface, and it is not shed into the circulation.4 As a
`consequence, PSMA can be considered a promising target for
`specific prostate cancer imaging and therapy.
`5−7
`The radiohalogenated inhibitors of PSMA exhibiting Glu-
`NH-CO-NH-Lys as a pharmacophore showed the ability to
`image PSMA-expressing prostate tumor xenografts.
`8,9 More
`recently, a corresponding DOTA-conjugate was labeled with
`68Ga which represents an attractive generator-based alternative
`to cyclotron-based PET radiopharmaceuticals.10,11 The readily
`available 68Ga decays with 89% probability by positron emission
`allowing high-resolution PET images with the option of
`accurate quantification. N,N′-Bis[2-hydroxy-5-(carboxyethyl)-
`benzyl]ethylenediamine-N,N′- diacetic acid (HBED-CC) was
`recently proposed as an efficient 68Ga chelator with fast
`complexing kinetics and a high in vitro as well as in vivo
`complex stability.12,13
`Besides the efficient Ga(III) complexing characteristics,
`HBED-CC was selected because of its lipophilic nature. It
`was found that the“active binding site” of PSMA is composed
`of two structural motifs, one representing a lipophilic pocket
`and the other interacting with urea-based inhibitors.
`14 A study
`comparing a series of linkers located between the urea-based
`motif and a
`99mTc chelator revealed a clear dependency of
`binding properties in favor of the more lipophilic compounds.14
`Further studies described t he binding site as a pocket
`interacting with the carboxylic groups and the zinc complexing
`urea on one hand and with hydrophobic, mostly aromatic
`groups on the other hand.
`15
`Received: June 1, 2011
`Revised: February 6, 2012
`Published: February 28, 2012
`Article
`pubs.acs.org/bc
`© 2012 American Chemical Society 688 dx.doi.org/10.1021/bc200279b | Bioconjugate Chem. 2012, 23, 688−697
`Downloaded via FURMAN DEMARIS on March 1, 2024 at 20:32:23 (UTC).
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`'V ACS Publications
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`This report describes the syntheses of Glu-NH-CO-NH-
`Lys(Ahx)-HBED-CC (7 ) and Glu-NH-CO-NH-Lys(Ahx)-
`DOTA ( 8) together with a reference PSMA inhibitor
`previously published by Banerjee et al.10 After 68Ga complex-
`ation, these compounds were evaluated in vitro and in vivo to
`study the influence of these two chelators and side chain
`variations.
`■ MATERIALS AND METHODS
`Reagents. All commercially available chemicals were of
`analytical grade and were used without further purification.
`68Ga was obtained from a 68Ge/68Ga generator based on
`pyrogallol resin support. 12,16 Protected amino acids were
`obtained from Novabiochem (Merck, Darmstadt, Germany)
`or IRIS Biotech (Marktredwitz, Germany).
`The preparations were analyzed using reversed-phase high-
`performance liquid chromatography (RP-HPLC; Chromolith
`RP-18e, 100 × 4.6 mm; Merck, Darmstadt, Germany). Runs
`were performed using a linear A−B gradient (0% B to 100% B
`in 6 min) at a flow rate of 4 mL/min. Solvent A consisted of
`0.1% aqueous TFA and solvent B was 0.1% TFA in CH
`3CN or
`MeOH (in case of determination of radiochemical yield (RCY)
`or analysis of serum stability).
`For all preparative purifications, a Chromolith RP-18e
`column (100 × 10 mm; Merck, Darmstadt, Germany) was
`used with a 6 min gradient starting at 0% raised to 50% and
`followed by a 1 min increase to 100% B. The flow rate was 6
`mL/min. The HPLC system (L6200 A; Merck-Hitachi,
`Darmstadt, Germany) was equipped with a variable UV and a
`gamma detector (Bioscan; Washington, USA). UV absorbances
`were measured at 214 and 254 nm. Mass spectrometry was
`performed with a MALDI-MS Daltonics Microflex (Bruker
`Daltonics, Bremen, Germany) system. High-resolution mass
`spectrometry was performed using a system equipped with a
`mass spectrometer supporting Orbitrap technology (Exactive,
`Thermo Fisher Scientific). Full-scan single mass spectra were
`obtained by scanning m/z = 200−4000. NMR data were
`obtained with Bruker Avance NMR Spectrometers, AV(I)-600
`(600 MHz) and AV(III)-400 (400 MHz). The chemical shifts
`are referenced to solvent signals (DMSO-d
`5 = 2.50/39.757
`ppm, DMSO-d6 = 39.50).
`Synthesis of Glu-NH-CO-NH-Lys(Ahx)-HBED-CC (7).In
`a first step, the isocyanate 2 of the glutamyl moiety was
`generated in situ by adding a mixture of 3 mmol of bis(tert-
`butyl) L-glutamate hydrochloride (Bachem, Switzerland) (1)
`and 1.5 mL ofN-ethyldiisopropylamine (DIPEA) in 200 mL of
`dry CH2Cl2 to a solution of 1 mmol triphosgene in 10 mL of
`dry CH2Cl2 at 0 °C over 4 h. After agitation of the reaction
`mixture for one further hour at 25°C, 0.5 mmol of a resin-
`immobilized (2-chloro-tritylresin, Merck, Darmstadt)ε-allylox-
`ycarbonyl protected lysine was added in one portion (in 4 mL
`DCM) and reacted for 16 h with gentle agitation leading to
`compound 3. The resin was filtered off and the allyloxy-
`protecting group was removed using 100 mg tetrakis-
`(triphenyl)palladium(0) (Sigma-Aldrich, Germany) and 400
`μL morpholine in 4 mL CH
`2Cl2 for 3 h resulting in compound
`4. The following coupling of the aminohexanoic moiety was
`performed using 2 mmol of the Fmoc-protected 6-amino-
`hexanoic acid (Sigma-Aldrich, Germany), 1.96 mmol of HBTU
`(Merck, Darmstadt, Germany), and 2 mmol of N-ethyl-
`diisopropylamine in a final volume of 4 mL DMF. The product
`5 was cleaved from the resin by reacting with 4 mL of a 30%
`1,1,1,3,3,3-hexafluoroisopropanole (HFIP) in CH
`2Cl2 for two
`hours at ambient temperature resulting in the tert-butyl
`protected crude product6 which was purified via RP-HPLC.
`To conjugate HBED-CC, the purified product6 was reacted
`with an equimolar amount of HBED-CC-TFP-ester synthesized
`as previously described
`12 in the presence of 2 equiv of DIPEA
`in N,N-dimethylformamide (DMF; final volume of 1 mL).
`After HPLC purification, the remainingtert-butyl groups were
`cleaved at room temperature for one hour using 2 mL
`trifluoroacetic acid to obtain7 in ∼35% yield after purification
`by HPLC. High-resolution mass spectrometry was used to
`confirm the identity (Table 1), and purity was analyzed via
`analytical HPLC at λ = 206 nm (Supporting Information).
`Complete and unequivocal NMR
`1H and 13C signal assign-
`ments were obtained (Supporting Information).
`Synthesis of Glu-NH-CO-NH-Lys(Ahx)-DOTA (8). Pre-
`cursor 5 was synthesized as mentioned before. After activation
`with 3.95 equiv of HBTU and DIPEA for 2 h, 4 equiv of tris(t-
`bu)-DOTA (Chematech, Dijon, France) relative to the resin
`loading were reacted with 5 after removal of the Fmoc-
`protecting group in a final volume of 3 mL DMF. The product
`was cleaved from the resin in a 2 mL mixture consisting of
`trifluoroacetic acid, triisopropylsilane, and water (95:2.5:2.5).
`The product was purified via semipreparative RP-HPLC
`resulting in compound8 (∼30% yield). High-resolution mass
`spectrometry was used to confirm the identity (Table 1) and
`purity was analyzed via analytical HPLC at λ = 206 nm
`(Supporting Information). Complete and unequivocal
`1H and
`13C NMR signal assignments were obtained (Supporting
`Information).
`Synthesis of Reference Compound (12). Synthesis of
`the DOTA conjugate previously published by Banerjee et al.10
`was performed using a modified solid-phase method. Five
`equivalents of suberic-acid-bis-( N-hydroxysuccinimideester)
`(Sigma-Aldich, Germany) was conjugated to the side chain of
`the lysine moiety of compound4 in the presence of 5 equiv of
`triethylamine in 3 mL DMF. After 2 h, the resin was washed
`with DMF. In the presence of five equiv of triethylamine, five
`equiv of Fmoc-Lys-oAll was coupled to the immobilized NHS-
`ester of intermediate9 in a final volume of 3 mL DMF for 16 h
`resulting in 10. The remaining conjugations of two phenyl-
`alanine building blocks and tris(t-bu)-DOTA were performed
`Table 1. Analytical and PSMA-Binding Data
`Ga-peptide
`complexes m/za
`m/z calculated as
`[M+H]+
`analytical HPLC
`retentionb
`affinity related IC50 [nM] determined in
`enzyme-based assayc
`affinity related Ki [nM] determined in
`cell-based assayc
`[Ga]7 947.4257 947.4250 3.1 min 7.5 ± 2.2 12.0 ± 2.8
`[Ga]8 819.4104 819.4100 2.4 min 19.4 ± 7.1 37.6 ± 14.3
`[Ga]12 1284.6368 1284.6364 3.7 min 8.7 ± 3.9 11.1 ± 1.8
`aHigh-resolution mass spectrometry data of the free ligands ([M+H]+). bCompounds were labeled with68Ga; runs were performed using a linear
`A−B gradient (0% B to 100% B in 6 min) at a flow rate of 4 mL/min. Solvent A was 0.1% aqueous TFA and solvent B was MeOH.cCompounds
`were complexed withnatGa.
`Bioconjugate Chemistry Article
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`according to standard Fmoc-protocols resulting in11. Cleavage
`from resin was performed using a 3 mL mixture of
`trifluoroacetic acid, triisopropylsilane, and water (95:2.5:2.5).
`The product was purified via RP-HPLC to obtain12 in ∼8%
`yield. High-resolution mass spectrometry was used to confirm
`the identity (Table 1), and purity was analyzed via analytical
`HPLC at λ = 206 nm (Supporting Information).
`68Ga-Labeling. Typically, 0.1−1 nmol of Glu-NH-CO-NH-
`Lys(Ahx)-HBED-CC (7, in 0.1 M HEPES buffer pH 7.5) or 1
`nmol of the DOTA conjugates (8/12, in 0.1 M HEPES buffer
`pH 7.5) were added in a volume of 100μL to a mixture of 10
`μL 2.1 M HEPES solution and 10μL[ 68Ga]Ga3+ eluate (50−
`100 MBq). The pH of the labeling solution was adjusted to 4.2.
`Depending on the chelator, the reaction mixture was incubated
`either at ambient temperature or at 80 °C for 2 min. The
`radiochemical yield (RCY) was determined using RP-HPLC.
`67Ga-Labeling. 67Ga was purchased from MDS Nordion
`(Fleurus, Belgium) as [67Ga]GaCl3 in 0.1 N HCl. Typically, 0.1
`nmol of Glu-NH-CO-NH-Lys(Ahx)-HBED-CC (7) was added
`in a volume of 100μL to a mixture of 10μL 2.1 M HEPES
`solution, 2μL 1 N HCl, and 10μL[ 67Ga]GaCl3 (∼100 MBq)
`in 0.1 N HCl resulting in a solution with a pH of 4.2. The
`reaction mixture was incubated for 2 min at ambient
`temperature. The RCY was determined using RP-HPLC.
`natGa-Complexes. A1 0× molar excess of Ga(III)-nitrate
`(Sigma Aldrich, Germany) in 0.1 N HCl (10μL) was reacted
`with the compounds7, 8,o r12 (1 mM in 0.1 M HEPES buffer
`pH 7.5, 40μL) in a mixture of 10μL 2.1 M HEPES solution
`and 2 μL 1 N HCl for 2 min at 80°C.
`Radiochemical Stability. The radiochemical stability of
`the 68Ga-labeled compounds 7, 8, and 12 was determined by
`incubating in both PBS and human serum for 2 h at 37°C. An
`equal volume of MeCN was added to the samples to precipitate
`serum proteins. Subsequently, the samples were centrifuged for
`5 min at 13 000 rpm. An aliquot of the supernatant and the PBS
`sample was analyzed by RP-HPLC. In addition, serum samples
`of compound [
`68Ga]7 were run on a Superdex 200 GL 5/150
`gel filtration column (GE Healthcare, Munich, Germany) to
`analyze protein binding. To analyze the complex stability
`against human transferrin, a 400 μL aliquot of [
`68Ga]7 was
`added to 250μg apo-transferrin in PBS at pH 7 and incubated
`at 37 °C (water bath) for 2 h. The complex stability was
`determined using a Superdex 200 GL 5/150 short column with
`PBS pH 7 as eluent.
`Naaladase Assay. Recombinant human PSMA (rhPSMA,
`R&D systems, Wiesbaden, Germany) was diluted in assay
`buffer (50 mM HEPES, 0.1 M NaCl, pH 7.5) to 0.4μg/mL.
`The substrate Ac-Asp-Glu (Sigma, Taufkirchen, Germany, 40
`μM final concentration) was mixed with [
`natGa]7,[ natGa]8,o r
`[natGa]12 at concentrations ranging from 0.05 nM to 1000 nM
`in a final volume of 125μL assay buffer. The mixtures were
`combined with 125μL of the rhPSMA solution (0.4μg/mL)
`and incubated for one hour at 37°C. The reaction was stopped
`by heating at 95°C for 5 min. 250μL of a 15 mM solution of
`ortho-phthaldialdehyde (Sigma, Taufkirchen, Germany) was
`added to all vials and incubated for 10 min at ambient
`temperature. Finally, 200μL aliquots of the reaction solutions
`were loaded onto a F16 Black Maxisorp Plate (Nunc,
`Langenselbold, Germany) and read at excitation and emission
`wavelengths of 330 and 450 nm, respectively, using a
`microplate reader (DTX-880, Beckman Coulter, Krefeld,
`Germany). The data were analyzed by a one-site total binding
`regression algorithm of GraphPad Prism (GraphPad Software,
`California, USA).
`Cell Binding Studies.Cell binding studies were performed
`using PSMA
`+ LNCaP cells (metastatic lesion of human
`prostatic adenocarcinoma, ATCC CRL-1740) and PSMA −
`PC-3 cells (bone metastasis of a grade IV prostatic
`adenocarcinoma, ATCC CRL-1435) cultured in DMEM
`medium supplemented with 10% fetal calf serum and 2
`mmol/L
`L-glutamine (all from Invitrogen). During cell culture,
`cells were grown at 37°C in an incubator with humidified air,
`equilibrated with 5% CO 2. The cells were harvested using
`trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA; 0.25%
`trypsin, 0.02% EDTA, all from Invitrogen).
`In order to determine the binding affinity, a competitive cell
`binding assay was performed. LNCaP cells (10
`5 per well) were
`incubated with a 0.2 nM solution of [67Ga]7 in the presence of
`12 different concentrations of [natGa]7,[ natGa]8,o r[ natGa]12
`(0−5000 nM, 200 μL/well). After incubation at ambient
`temperature for 1 h with gentle agitation, the binding buffer
`was removed using a multiscreen vacuum manifold (Millipore,
`Billerica, MA). After washing twice with 100μL and once with
`200 μL ice-cold binding buffer, the cell-bound radioactivity was
`measured with a gamma counter (Packard Cobra II, GMI,
`Minnesota, USA). The 50% inhibitory concentration (IC
`50)
`values were calculated by fitting the data using a nonlinear
`regression algorithm (GraphPad Software). Experiments were
`performed at least three times including quadruplicate sample
`measurements.
`Determination of Binding Specificity and Internal-
`ization. Internalization experiments were performed as
`previously described.
`17 Briefly, 105 LNCaP or PC-3 cells were
`seeded in poly(L-lysine)-coated 24-well cell culture plates 24 h
`before incubation. After washing with PBS, the cells were
`incubated with the radiolabeled compounds [
`68Ga]7,[ 68Ga]8,
`and [68Ga]12 (25 nM final concentration) for 45 min at 37°C
`and at 4 °C, respectively. To determine specific cell uptake,
`cells were blocked with 2-(phosphonomethyl)-pentanedioic
`acid (PMPA, Axxora, Loerrach, Germany) to a final
`concentration of 100 μM. Cellular uptake was terminated by
`washing 4 times with 1 mL of ice-cold PBS. To remove surface-
`bound radioactivity, cells were incubated twice with 0.5 mL
`glycine-HCl in PBS (50 mM, pH 2.8) for 5 min at room
`temperature. The cells were washed with 1 mL of ice-cold PBS
`and lysed using 0.5 mL of 0.3 M NaOH. The surface-bound
`and internalized fractions were measured in a gamma counter.
`Specificity of binding was additionally analyzed in a
`concentration-dependent cell uptake experiment. Solutions of
`[
`68Ga]7,[ 68Ga]8,o r[68Ga]12 at final concentrations of 2.5, 25,
`and 250 nM were added to 7× 105 cells suspended in 50μL
`OPTIMEM medium (Gibco, Auckland, New Zealand). After
`45 min at 37°C, the samples were briefly vortexed and a 10μL
`aliquot was transferred to a 400μL microcentrifuge tube (Roth,
`Germany) containing 350μL of a 75:25 mixture of silicon oil,
`density 1.05 (Sigma Aldrich, Germany), and mineral oil, density
`0.872 (Acros, Nidderau, Germany). Separation of cells from the
`medium was performed by centrifugation at 12 000 rpm for 2
`min. After freezing the tubes using liquid nitrogen, the bottom
`tips containing the cell pellet were cut off. The cell pellets and
`the supernatants were separately counted in a gamma counter.
`Biodistribution and PET Imaging. Five ×10
`6 cells of
`either LNCaP or PC-3 in 50% Matrigel (Becton Dickinson,
`Heidelberg, Germany) were subcutaneously inoculated into the
`right trunk of male 7- to 8-week-old BALB/c nu/nu mice
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`(Charles River Laboratories). The tumors were allowed to grow
`for 3 to 4 weeks until approximately 1 cm3 in size.
`The 68Ga-radiolabeled compounds of 7, 8, and 12 were
`injected via tail vein (1−2 MBq per mouse; 0.1−0.2 nmol). At
`1 h after injection, the animals were sacrificed. Organs of
`interest were dissected, blotted dry, and weighed. The
`radioactivity was measured with a gamma counter and
`calculated as % ID/g.
`For the microPET studies, 10 −25 MBq of compounds
`[
`68Ga]7 and [68Ga]8 in a volume of 0.15 mL (∼0.5 nmol) were
`injected via a lateral tail vein into mice bearing LNCaP tumor
`xenografts. The anesthetized animals (2% sevoflurane, Abbott,
`Wiesbaden, Germany) were placed in prone position into the
`Inveon small animal PET scanner (Siemens, Knoxville, Tenn,
`USA) to perform a 50 min dynamic microPET scan starting at
`1 min post injection followed by a 20 min static scan.
`Statistical Aspects. All experiments were performed at
`least in triplicate. Quantitative data were expressed as mean±
`SD. If applicable, means were compared using Student’s t-test.
`P values of <0.05 were considered statistically significant.
`■ RESULTS
`Chemistry. The synthesis of the resin-bound intermediate5
`was performed using solid-phase chemistry as outlined in
`Scheme 1. To couple the respective chelators, compound5 was
`either cleaved from resin and reacted with an equimolar
`amount of HBED-CC-TFP-ester resulting in7 or coupled with
`HBTU activated tris(t-bu)DOTA resulting in the DOTA-
`conjugate 8. The preparation of the reference compound12
`previously published by Banerjee et al.
`10 was performed using a
`modified solid-phase method as outlined in Scheme 2. Briefly,
`disuccinimidyl suberate was conjugated to theε-amino group of
`the lysine group of4. After Fmoc-Lys-OAll was coupled to the
`resulting immobilized NHS-ester, the conjugation of two
`phenylalanine building blocks and tris( t-bu)-DOTA were
`performed according to standard Fmoc-protocols. Analytical
`data of compounds7, 8, and 12 are summarized in Table 1.
`Radiolabeling and Stability.Radiolabeling with
`68Ga was
`performed at pH 4.2 by incubating the conjugates7, 8,o r12 in
`a mixture consisting of 50−100 MBq [68Ga]Ga3+ and HEPES.
`Radiochemical yields were determined by RP-HPLC analysis of
`the reaction mixtures. The retention times obtained for each
`compound are shown in Table 1. An amount of 0.1 nmol of the
`Scheme 1. Syntheses of Glu-NH-CO-NH-Lys(Ahx)-HBED-CC (7) and Glu-NH-CO-NH-Lys(Ahx)-DOTA (8)
`(a) Triphosgene, DIPEA, CH2Cl2,0 °C; (b) H-Lys(Alloc)-2CT-Resin, CH2Cl2; (c) Pd[P(C6H5)3]4, morpholine, CH2Cl2; (d) Fmoc-6-Ahx-OH,
`HBTU, DIPEA, DMF; (e) 20% piperidine, DMF; (f) hexafluoroisopropanol/CH2Cl2; (g) HBED-CC-TFP ester, DIPEA, DMF; (h) TFA; (i) tris(t-
`bu)DOTA, HBTU, DIPEA; (j) TFA.
`Bioconjugate Chemistry Article
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`HBED-CC conjugate 7 in a final concentration of 1.7μM led
`to a radiochemical yield of more than 99% in less than 1 min at
`room temperature. As a consequence, specific activities in the
`range 500−1000 GBq/μmol were obtained. In order to achieve
`comparable high radiochemical yields with the DOTA-
`conjugates 8 and 12, the compounds were incubated for 2
`min at 80°C using 1 nmol precursor.
`Incubation of the 68Ga-labeled compounds 7, 8, and 12 in
`human serum for 2 h at 37°C resulted in no detectable changes
`in the radiograms. Stability of the 68Ga-labeled HBED-CC-
`Scheme 2. Modified Solid-Phase Synthesis of the DOTA Conjugate Previously Published by Banerjee et al.10
`(a) Disuccinimidyl suberate (DSS), TEA, DMF; (b) Fmoc-Lys-oAll, TEA, DMF; (c) 20% piperidine, DMF; (d) Fmoc-Phe-OH, HBTU, DIPEA,
`DMF; (e) tris(t-bu)DOTA, HBTU, DMF; (f) Pd[P(C6H5)3]4, morpholine, CH2Cl2; (g) TFA, triisopropylsilane, H2O (95/2.5/2.5).
`Figure 1. Determination of binding affinity of compounds [natGa]7,[ natGa]8, and [natGa]12 by competitive titration on LNCaP cells (A) and
`purified receptor using an enzyme-based assay (B). Data are expressed as mean± SD (n = 4). cpm = counts per minute; FI = fluorescence intensity.
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`Petitioner GE Healthcare – Ex. 1030, p. 692
`b
`-------
`A
`1500000
`E 1000000
`fr
`500000
`4
`• [natGa]7
`'" [natGa]8
`♦ [natGa]12
`104
`9
`B
`800 • [natGa]7
`.. [natGa]S
`600 ♦ [natGa]12
`u:::
`400
`200
`10 -1 100 10 1 103
`C [nM]
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`conjugate 7 could additionally be demonstrated by transferrin
`challenging and incubation in human serum. No activity was
`exchanged in presence of 250μg transferrin or transferred to
`other serum proteins.
`Determination of PSMA Affinity. The variants were
`competitively analyzed in terms of their binding capacity by
`performing an enzyme-based assay on rhPSMA (Naaladase-
`Assay) and a binding assay on LNCaP cells with
`67Ga-labeled 7
`(RCY > 99%; specific activity ∼1000 GBq/μmol) as radio-
`ligand. The affinity related IC50 and the calculatedKi values of
`both assays were determined by analyzing the respective
`binding curves (Figure 1) withGraphPad Prism. The data are
`summarized in Table 1. Compared to the DOTA conjugate
`[
`natGa]8, the HBED-CC conjugate [ natGa]7 and the more
`lipophilic reference compound [natGa]12 showed slightly better
`affinities to both the purified extracellular receptor rhPSMA
`and LNCaP cells.
`Specific Cell Binding and Internalization.Comparative
`cell uptake and internalization experiments with LNCaP
`(PSMA positive) and PC-3 (PSMA negative) cells revealed
`unspecific cell uptake of [
`68Ga]8 and [68Ga]12 in both cell lines
`which could not be blocked with excess PMPA (Figure 2A). In
`contrast, the HBED-CC conjugate ([68Ga]7) showed almost
`no uptake in PSMA-negative PC-3 cells or in LNCaP cells
`blocked with PMPA. Furthermore, [ 68Ga]7 significantly
`revealed higher specific internalization and cell surface
`accumulation as compared to the DOTA conjugates (Figure
`2B). In another cell uptake experiment, three different
`concentrations of the radiolabeled compounds (2.5 nM, 25
`nM, and 250 nM) were incubated with either LNCaP (Figure
`3A) or PC-3 cells (Figure 3B). In accordance with the results
`received from the internalization experiments, [
`68Ga]7 showed
`a considerably higher uptake in LNCaP cells. Furthermore, the
`unspecific uptake in PC-3 cells was lower as compared to the
`DOTA derivatives [
`68Ga]8 and [68Ga]12.
`Biodistribution Studies. The organ and tumor uptake
`values of the complexes [ 68Ga]7,[ 68Ga]8, and [ 68Ga]12
`differed in accordance with the aforementioned in vitro data
`(Figure 4). The HBED-CC conjugated compound [68Ga]7 was
`cleared rapidly from the circulation and PSMA negative tissue.
`Remarkably, the liver activity of [
`68Ga]7 amounted to only 0.87
`± 0.05% ID/g as early as one hour after injection. The high
`kidney, spleen, and lung uptake of 139.4± 21.4% ID/g, 17.90
`± 2.87% ID/g, and 2.49± 0.27% ID/g was nearly completely
`blocked to 4.02± 1.14% ID/g, 1.54± 0.33% ID/g, and 0.64±
`0.32% ID/g, respectively, after the coinjection of 2 mg/kg 2-
`PMPA (Figure 4B). The tumor uptake amounted to 7.70±
`1.45% ID/g on LNCaP and 1.30± 0.12% ID/g on PC-3.
`The DOTA complex [
`68Ga]8 showed a completely different
`uptake pattern. The liver uptake of the DOTA-conjugate was
`enhanced by a factor of 5.7, the tumor uptake was reduced by
`factor 2.6, and the kidney uptake was surprisingly lowered to
`background values. The uptake values of PSMA-negative PC-3
`tumors were low for all compounds (1.30 ± 0.13% ID/g
`([
`68Ga]7), 0.60 ± 0.06% ID/g ([68Ga]8), and 0.58 ± 0.07%
`ID/g ([68Ga]12), respectively) (Figure 4C).
`PET-Imaging. The tumor-targeting efficiency of [68Ga]7
`and [68Ga]8 was evaluated by 50 min dynamic microPET scans
`followed by a 20 min static scan. The differences observed in
`biodistribution could have also been visualized by PET imaging.
`The complexes [
`68Ga]7 and [ 68Ga]8 were injected intra-
`venously into LNCaP tumor-bearing mice, and microPET scans
`were carried out for up to 70 min p.i. A coronal PET slice after
`injection of [
`68Ga]7 is shown in Figure 5a. The DOTA complex
`[68Ga]8 failed to image the tumor proven by the time activity
`curves over tumor and muscle (Figure 5B).
`Comparison of Biological Properties of L- and D-
`Forms of [68Ga]7. The 68Ga complex ofD-Glu-NH-CO-NH-
`Lys(Ahx)-HBED-CC was synthesized and compared with the
`corresponding L-isomer. Time−activity curves obtained from
`dynamic PET measurements revealedD-[68Ga]7 to be cleared
`from the kidneys into the urinary bladder whileL-[68Ga]7 is
`retained in this organ (Figure 6). This finding is supported by
`the ∼10
`3 times lower PSMA affinity of D-[68Ga]7 (data not
`shown).
`■ DISCUSSION
`PSMA is strongly expressed on prostate cancer cells 18 and
`therefore represents a promising target for the development of
`imaging agents for tumor staging and followup. Among the
`currently used radioisotopes,
`68Ga exhibits several favorable
`properties as there are the availability and the facile labeling
`technology by means of complexation.
`11 A series of chelators
`are available for the complexation of68Ga which are conjugated
`to targeting carrier molecules. Besides some reported stabilizing
`Figure 2. Cell binding and internalization of compounds [68Ga]7,
`[68Ga]8, and [68Ga]12 (A). Specific cell uptake was evaluated by
`blockade with 100 μM PMPA. Specificity of cell uptake (B) was
`calculated by subtracting the respective signals resulting from PMPA-
`blockage. Values are expressed as % of applied radioactivity bound to
`106 cells. Data are expressed as mean± SD (n = 3).
`Bioconjugate Chemistry Article
`dx.doi.org/10.1021/bc200279b | Bioconjugate Chem. 2012, 23, 688−697693
`Petitioner GE Healthcare – Ex. 1030, p. 693
`-----------------------------
`A
`C.
`ra
`u 10
`z
`...J
`C:
`0
`Cl
`.5 5
`"C
`C:
`:ci
`'?F-
`0 u u <( <( u
`~ ~ a. a. ~ .., :. :. ..,
`a. a.
`+ +
`p u
`.. ~ ..,
`[6aGa]7
`B
`10
`C. .. 8 (.)
`z
`...J
`C: 6
`0
`Cl)
`C: 4
`'ti
`C:
`:a 2
`'?F-
`0 u u
`~ ~ ..,
`u <( <(
`~ a. a.
`:. :. a. a.
`+ +
`p u
`.. ~ ..,
`[68Ga]8
`u u
`~ ~ ..,
`u u <( <(
`~ ~ a. a. .., :. :. a. a.
`+ +
`p u
`.. ~ ..,
`[68Ga]12
`u u
`~ ~ ..,
`D 1st glycine \/"lash
`■ 2nd glycine \/"lash
`• Lysate
`□ 1st glycine wash
`■ 2nd glycine wash
`-
`Lysate
`
`
`
`
`
`
`
`effects or pretargeting approaches, 17,19 the function of the
`chelator in radiopharmaceuticals is usually restricted to serving
`as a complexing agent without influencing the receptor binding.
`However, the size-demanding radiometal complexes often exert
`their influence on the targe ting molecule by changing
`lipophilicity or charge. In particular, the pharmacological
`property of small molecules can strongly be influenced or
`abolished with respect to its target binding properties.
`An interesting study comparing various linkers located
`between the PSMA binding group 2-[3-(1,3-dicarboxyprop-
`yl)-ureido]pentanedioic acid (DUPA) and a
`99mTc complex
`showed that the lipophilicity of the linker significantly
`correlates with improved binding properties.14 Crystal structure
`investigations also indicated that, besides the electrostatic
`interactions of urea and carboxylic groups at the active, Zn(II)-
`containing center of PSMA, there are lipophilic interactions
`resulting from a hydrophobic pocket located next to the active
`site.
`8,20 A further study substantiates the PSMA active site as a
`pocket with multiple interactions as well. The pharmacophore
`was proposed to present three carboxylic groups able to interact
`with the respective side chains of PSMA, an oxygen as part of
`zinc complexation in the active center and an aromatic structure
`able to interact with a hydrophobic part of the binding pocket
`composed of tyrosines.
`15 This work suggests that these
`interactions are additive in terms of functional efficiency.
`Only inhibitors which interact with all parts of the binding site
`displayed a tight binding mode and a high rate of internal-
`Figure 3. Binding of [68Ga]7,[ 68Ga]8, and [68Ga]12 to LNCaP (PSMA+) cells (A) and PC-3 (PSMA−) cells (B) depending on68Ga-complex
`concentration. Values were normalized with respect to added radioactivity of each compound. Data are expressed as mean± SD (n = 3).
`Figure 4.Organ distribution expressed as % ID/g tissue one hour post injection. (A) Comparison of the compounds [68Ga]7 (PSMA-binding motif
`coupled to HBED-CC), [68Ga]8 (PSMA-binding motif coupled to DOTA), and [68Ga]12. (B) PSMA-blocking by coadministration of 2 mg/kg
`body weight PMPA indicating PSMA-specific uptake in kidney and spleen. (C) Uptake in PC-3 tumors and LNCaP tumors 1 h after injection. Data
`are expressed as mean± SD (n = 3).
`Figure 5. Whole-body coronal microPET image of an athymic male nude mice bearing LNCaP tumor xenografts. The tumor-targeting efficacy of
`[68Ga]7 was demonstrated by dynamic microPET scan (B) followed by a static scan (A). The static scan up to 1 h post injection of [68Ga]7 is shown
`in (A). Approximately 15 MBq/mouse was injected. Graph B shows the respective time−activity curves in muscle and tumor for [68Ga]7 and [68Ga]
`8.
`Bioconjugate Chemistry Article
`dx.doi.org/10.1021/bc200279b | Bioconjugate Chem. 2012, 23, 688−697694
`Petitioner GE Healthcare – Ex. 1030, p. 694
`A =
`60000 LNCaP I!!!!!!!
`-
`40000
`E
`C.
`u
`20000
`0
`[68 Ga)7 [68Ga)8 [68Ga)12
`D [68Ga]7
`- [68 Ga ]8
`- [68 Ga ]12
`2.5nM 8
`25 nM
`250 nM
`E
`C.
`u
`B
`4
`60000
`40000
`20000
`0
`[68Ga)7
`D [68 Ga ]7
`- [68G a]7 +
`2 mg/kg PM PA
`C 10
`9
`8
`7
`~ 6
`C 5
`~ 4
`3
`2
`PC-3
`[68 Ga)8 [68 Ga)12
`1
`o -'--J____u_._._ ,__--1_.1..
`=
`11!!!!1
`--2.5 nM
`25 nM
`250 nM
`D [68 Ga]7
`- [68 Ga]8
`lililil [68 Ga ]12
`P C-3 Tum or LNCa P Tumo r
`-+- Muscle
`-+- Tumor
`-+- Muscle
`-+- Tumor
`0-,...------.--------r-----,
`0 20 40 60
`time after injection [min]
`
`
`
`
`
`
`
`ization. Molecules lacking one of these interactions showed
`other modes of action and weaker binding.15
`These results led to the development of the amphiphilic,
`PSMA-specific tracer [ 68Ga]7 consisting of the urea-based
`pharmacophore and the 68Ga-HBED-CC complex able to
`interact with the hydrophobic binding pocket. Two 68Ga-
`DOTA complexes, the hydrophilic [68Ga]8