Conjugated to Cyclic RGD Peptides - American Chemical Society

Nov 11, 2010 - Oslo, Norway, Centre for Molecular Biology and Neuroscience (CMBN), University of Oslo, ... Revised Manuscript Received October 21, 201...
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Bioconjugate Chem. 2010, 21, 2297–2304

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Radiosynthesis and Biodistribution of a Prosthetic Group (18F-FENMA) Conjugated to Cyclic RGD Peptides Dag Erlend Olberg,*,† Alan Cuthbertson,‡ Magne Solbakken,‡ Joseph M. Arukwe,‡ Hong Qu,§ Alexandr Kristian,| Skjalg Bruheim,| and Ole Kristian Hjelstuen†,‡ Department of Pharmaceutics & Biopharmaceutics, University of Tromsø, Norway, GE Healthcare Medical Diagnostics R&D, Oslo, Norway, Centre for Molecular Biology and Neuroscience (CMBN), University of Oslo, Norway, and Department of Tumour Biology, Institute of Cancer Research, Rikshospitalet, Oslo, Norway. Received July 16, 2010; Revised Manuscript Received October 21, 2010

We have recently reported a new N-methylaminooxy-based prosthetic group for the site-selective introduction of 18 F-fluorine under mild acidic aqueous conditions into model peptides functionalized with a Michael acceptor moiety. To further investigate the utility of this methodology, the radiosynthesis of two cyclic RGD peptides was carried out, and in vivo biodistribution and microPET studies were performed in tumor-bearing mice. A cyclic RGD peptide was functionalized with the Michael acceptors trans-β-nitrostyrene carboxylic acid and 3-vinylsulfonylpropionic acid. Radiolabeling was then performed with the prosthetic group O-(2-(2-[18F]fluoroethoxy)ethyl)-N-methylhydroxylamine (18F-FENMA) yielding the 18F-conjugates in moderate yields (8.5-12%). Biodistribution, blocking, and microPET imaging studies were performed in a mouse xenograft model. The vinylsulfonyl-modified conjugate demonstrated good in vitro plasma stability. Biodistribution and microPET studies revealed excellent tumor uptake with low background in key organs and renal elimination as the predominant route of excretion. Blocking studies with coinjected nonlabeled RGD peptide confirmed the in vivo specificity for the integrin Rvβ3. On the other hand, 18F-FENMA-nitrostyrene-RGD, although stable at conjugation pH 5, was found to rapidly degrade at physiological pH through loss of the 18F-prosthetic group.

INTRODUCTION Positron emission tomography (PET) has become an important functional imaging modality in diagnostic medicine due to the high sensitivity, high resolution, and ability to quantify the radioactive concentration in tissues (1). Fluorine-18 is a particularly attractive radionuclide due to the favorable nuclear and chemical properties (2), as well as its availability from medical cyclotrons in high specific activity (3, 4). The low positron energy of 0.64 MeV results in low radiation doses and short tissue range, and the relatively long 109.7 min half-life allows both multistep radiosynthesis and extended scanning protocols (5). Although numerous drug-like molecules have been successfully labeled with fluorine-18, optimization of the radiolabeling route to complex molecules such as peptides and proteins still offers significant challenges to the radiochemist. Direct incorporation of [18F]fluoride at high specific radioactivity into macromolecules is hampered by the harsh reaction conditions, and as a consequence, peptide and protein labeling is usually carried out by adopting a prosthetic group strategy (6). This methodology, also referred to as radiolabeling using bifunctional labeling agents, involves first 18F incorporation into a small organic molecule, which is then conjugated to the macromolecule under mild conditions in a second step. The variety of chemistries exploited for the conjugations includes acylation, Huisgen 1,3-dipolar cycloaddition (click chemistry), alkylation, and oxime or hydrazone forming reactions (7-10). Each * Corresponding author. Dag Erlend Olberg; e-mail: dag.erlend. [email protected]. Phone: + 47 230 76327. Fax: +47 230 76310. † University of Tromsø. ‡ GE Healthcare Medical Diagnostics R&D. § University of Oslo. | Institute of Cancer Research.

approach has its own advantages and limitations affecting the radiolabeling yields and biodistribution profile of the tracer. For example, increasing the lipophilicity of the prosthetic group changes the physicochemical properties of the drug product leading to increased hepatobiliary excretion (11). In order to control excretion routes, peptides are often modified with poly(ethylene glycol) (PEG) units or other pharmacokinetic modifiers (PKMs) (12, 13) to counteract the lipophilic nature of the prosthetic group. As an alternative, hydrophilic prosthetic groups are therefore considered desirable. Recently, we reported a novel prosthetic group O-(2-(2[18F]fluoroethoxy)ethyl)-N-methylhydroxylamine (18F-FENMA) utilizing the site-selective reactivity of the N-methylaminooxy functionalitywithpeptidesmodifiedwithaMichaelacceptor(14,15). Further extension of this approach is reported in this study using the RGD peptide 1 modified with either 4-[(E)-2-nitrovinyl]benzoic acid or a 3-vinylsulfonylpropionyl group. The nitrostyrene-peptide 2 showed rapid reaction kinetics with 18FFENMA forming a more hydrophobic peptide as evidenced by radio-HPLC. The 3-vinylsulfonylpropionyl-peptide 3 formed the more hydrophilic conjugate [18F]5 but with slower reaction kinetics. The ultimate aim of this study was to determine the radiochemical yields, in vitro stability, biodistribution, and tumor targeting properties of the peptide conjugates in osteosarcoma xenograft bearing mice.

EXPERIMENTAL PROCEDURES Materials. All chemicals were of analytical grade and obtained commercially. No carrier added [18F]F- was produced via the 18O(p,n)18F reaction by bombardment of an isotopically enriched [18O]water target with 16.5 MeV protons using a GE PETtrace 6 cyclotron (Uppsala, Sweden). NMR spectra were run on a Bruker Avance III 400 spectrometer (Fa¨llanden, Switzerland) equipped with a 5 mm BBFO PFG probe.

10.1021/bc1003229  2010 American Chemical Society Published on Web 11/11/2010

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Preparative reversed-phase HPLC was performed on a Shimadzu LC-8A (Bergman, Oslo, Norway) system using a Phenomenex Luna C18(2) column (Teknolab, Oslo, Norway) (250 mm × 21.2 mm, 5 µm), flow rate 10 mL/min, with gradients 0-30% solvent B (Preparative Method 1) or 0-40% solvent B (Preparative Method 2) over 60 min (solvent A, water/0.1% TFA; solvent B, acetonitrile/0.1% TFA). LC-MS spectra were recorded on a LCQ DECA XP MAX instrument using electrospray ionization (ESI) or a Waters LCT-TOF (Instrument teknikk AS, Oslo, Norway) instrument (for high-resolution mass spectrometry (HRMS)) using a Phenomenex Luna (Teknolab) C18(2) column (20 mm × 2 mm, 3 µm), flow rate 0.6 mL/min with gradient 0-30% B. UV detection was in all instances at 214 and 254 nm. Synthesis of the tosylate precursor and labeling with [18F]fluoride yielding O-(2-(2-[18F]fluoroethoxy)ethyl)-Nmethylhydroxylamine (abbreviated 18F-FENMA) was carried out as described previously (14). The cyclic RGD peptide NC100717, was supplied by GE Healthcare (Oslo, Norway) (16). Bocprotected cysteic acid (N-Boc-L-cysteic acid) was prepared according to Jia et al. (17). Chemistry and Radiochemistry. Synthesis of Peptide (1). To a stirred solution of NC100717 (100 mg, 97 µmol) in DMF (3 mL) was added Boc-L-cysteic acid (32 mg, 115 µmol) preactivated with tetramethylfluoroformamidiniun hexafluorophosphate (TFFH) (31 mg, 115 µmol) and N,N-diisopropylethylamine (DIPEA) (101 µL, 0.58 mmol). After 2 h, solvent was removed in vacuo, and the crude residue was treated with trifluoroacetic acid (TFA) (5 mL) for 5 min. TFA was removed under reduced pressure. The two-step cycle described above was repeated to introduce and deprotect the second cysteic acid residue. The crude mixture was then subjected to purification by preparative HPLC (Method 1), and after removal of the organic solvents under reduced pressure, the pure product (>95%) was isolated by lyophilization. Yield: 26 mg (17% based on NC100717). LC-MS found m/z ) 1560.5 [M+H]+ calculated for C56H89N17O25S5 1559.48. Synthesis of 3-(Vinylsulfonyl)propionic Acid. To a stirred solution of 3-(2-chloro-ethanesulfonyl)propionic acid methyl ester (100 mg) in dichloromethane (5 mL) was added 3 equiv of triethylamine at room temperature, and the reaction mixture was allowed to stand overnight. After removal of solvents under reduced pressure, the residue was purified by normal-phase flash chromatography (gradient 0-50% ethyl acetate in hexane). Purified intermediate was dissolved in 0.1 M HCl (20 mL) and refluxed overnight. The aqueous phase was evaporated off, and the residue was dissolved in dichloromethane and dried over Na2SO4 for 1 h. After filtration and removal of solvents, 3-(vinylsulfonyl)propionic acid was obtained as a white powder (59 mg, 77%). 1H NMR (400 MHz, CDCl3): δ (ppm) 6.65 (dd, J1 ) 9.7 Hz, J2 ) 16.7 Hz, 1H), δ 6.49 (d, J ) 16.7 Hz, 1H), δ 6.22 (d, J ) 9.7 Hz, 1H), δ 3.34 (t, J ) 7.4 Hz, 2H), δ 2.87 (t, J ) 7.4 Hz, 2H). 13C NMR (100 MHz, CDCl3): δ (ppm) 173.8, 135.8, 131.5, 49.2, 27.0. Synthesis of Nitrostyrene-RGD (2) and Sulfonyl-RGD (3). 4-[(E)-2-Nitrovinyl]benzoic acid was synthesized as previously described (18). To a stirred solution of 1 (13 mg, 7.9 µmol) in DMF (3 mL) was added either 4-[(E)-2-nitrovinyl]benzoic acid or 3-vinylsulfonylpropionic acid (12 µmol) preactivated with TFFH (3.2 mg, 12 µmol) and DIPEA (10 µL, 0.60 µmol). The reaction mixtures were placed in a sonication bath for 1 h and product formation was monitored by LC-MS. If necessary, the above procedure was repeated until complete conversion was achieved. Reaction mixtures were subjected to purification by preparative HPLC (method 2 compound 2, method 1 compound 3), and products were isolated from fractions by lyophilization. Nitrostyrene-RGD (2): Yield 8.2 mg (60%), LC-MS found m/z ) 1735.7, [M+H]+ calculated for C65H94N18O28S5 was 1734.5.

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Sulfonyl-RGD (3): Yield 7.4 mg (55%), LC-MS found m/z ) 1706.7, [M+H]+ calculated for C61H95N17O28S6 was 1705.5. Synthesis of NonradioactiVe Reference Standard FENMA Conjugates 4 and 5. To a solution of O-(2-(2-fluoroethoxy)ethyl)-N-methylhydroxylamine (1 mg, 4 µmol) in acetate buffer 0.4 M pH 5 (1 mL) was added either 2 or 3 (0.9 µmol). Conversion to 4 was completed after only 15 min at room temperature, whereas 5 was formed in >90% yield with heating to 60 °C for 60 min. Reaction mixtures were subjected to purification by preparative HPLC (method 2), and the pure products were isolated from fractions by lyophilization. (4): Yield 1.4 mg (85%), LC-HRMS found m/z ) 1870.5868, [M-H]- calculated for C70H106FN19O30S5 was 1871.5941. (5): Yield 1.3 mg (80%), LC-HRMS found m/z ) 1841.5627, [M-H]- calculated for C66H107FN18O30S6 was 1842.5709. Radiosyntheses of [18F]4 and [18F]5. Peptide precursors 2 and 3 were radiolabeled by reaction with 18F-FENMA in acetate buffer pH 5 with a specific activity of 40-50 GBq/µmol (after formulation) as determined by comparison of the radiochromatogram with a calibration curve of authentic reference standards. The radiosynthesis and purification of 18F-FENMA was carried out as previously described (14). In short, a solution of 2 or 3 (1.2 µmol) in acetate buffer 0.4 M pH 5 (0.8 mL) was added to the reaction vial containing purified 18F-FENMA. Conjugation conditions were 40 °C for 15 min and 70 °C for 60 min for [18F]4 and [18F]5, respectively. Postconjugation, the reaction mixture was diluted with water (4 mL) before purification by C18 reversed-phase liquid chromatography using both radiometric detection and UV detection at 214 nm. The HPLC fraction containing the radioactive material was diluted with water and passed through a Waters C18 Sep-Pak light cartridge preactivated with ethanol (2 mL) and water (5 mL). The cartridge with bound drug product was washed with water (5 mL), and the product was eluted into a sealed sterile vial with 1 mL absolute ethanol. The ethanol was removed under a stream of nitrogen at 70 °C. The radioactive residue was reconstituted in isotonic saline (adjusted to pH 4 for compound [18F]4) for use in the animal experiments. The identities of [18F]4 and [18F]5 were confirmed by coelution with the corresponding nonradioactive reference compounds. Integrin Receptor-Binding Assay. The binding affinity and specificity of 4 and 5 for Rvβ3 integrin were assessed in a competitive cell binding assay using 125I-echistatin and membrane preparations from the human endothelial adenocarcinoma cell line EA-Hy926 as previously described (16). Ki values were calculated from the binding curves using Graphpad Prism software. In Vitro Stability in Mice Plasma. Approximately 2 MBq each of either [18F]4 and [18F]5 in saline (50 µL) was added to freshly collected mouse plasma (0.5 mL total volume) and the samples incubated at 37 °C. After 30 min, 1 and 2 h aliquots (100 µL) were collected, diluted 1:1 with phosphate buffered saline (PBS), and the mixtures filtered (Ultrafree filter unit 5000 NMWL, Millipore) at 14 000 rpm for 15 min. The resulting filtrate was diluted with water/0.1% TFA and analyzed by C18 reversedphase radio-HPLC. Octanol/Water Partition Coefficient. Approximately 50 kBq of [18F]5 in 30 µL PBS was diluted with 470 µL of PBS and added to 500 µL of n-octanol in an Eppendorf cup (n ) 4). After vortexing for 3 min, the cups were centrifuged (10 300 rpm; 6 min), and the radioactivity of 100 µL aliquots of the PBS and n-octanol phases was counted in a gamma counter. Animals and Tumor Model. Housing and handling of animals were performed according to protocols approved by the Animal Care and Use Committee, in compliance with the National Committee for Animal Experiments guidelines on animal welfare. Female Balb/c nu/nu mice, bred at the

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nude rodent facility at the Norwegian Radium Hospital, were maintained under specific pathogen-free conditions with food and water supplied ad libitum. The xenografts have previously been established by direct implantation of patient tumor specimens in nude mice (19). The xenografts were established by subcutaneous injection of human osteosarcoma cells (3 × 106) in 100 µL of PBS solution into distal part of mouse right flank when animals were 4-6 weeks of age. When the average tumor diameter was about 5-10 mm (14-21 days after implant), the mice underwent biodistribution and microPET imaging experiments. Visualization of neovascularization in the tumor tissue was performed by immunohistochemistry with a rat antimouse-CD31 monoclonal antibody (clone MEC 13.3; BD Pharmigen). Biodistribution and MicroPET Studies. Biodistribution. Nude mice bearing subcutaneously xenografted human OHS tumors were injected intravenously with approximately 1 MBq of [18F]5. Animals were euthanized and sacrificed at 5 min and 2 h post-injection (3 or 4 per time point). Blood, tumor, and the major organs and tissues were collected, wet-weighed, and their radioactivity counted in a gamma counter (MINAXI 5000 series, United Technologies Packard, Zurich, Switzerland). The percentage of injected dose per gram (%ID/g) was calculated for each sample. For each mouse, the amount of radioactivity present in the tissue samples was calibrated against a calibration curve of the injectate. Values are expressed as mean ( standard deviation (SD) for a group of 3-4 animals. For the receptor blocking experiment, [18F]5 was coinjected with 10 mg/kg of NC100717 in tumor bearing mice. Biodistribution was determined as described above at 2 h post-injection. MicroPET. PET scans and image analysis were performed using a microPET F120 scanner (Siemens Medical Solutions). Tumor-bearing mice were injected via a tail vein with 4-7 MBq of [18F]5. Static PET images were acquired for 15 min in mice under isoflurane anesthesia 105 min post-injection. For dynamic PET scans, mice were prepositioned in the microPET scanner under isoflurane anesthesia, injected with tracers, and imaged continuously from time 0 to 120 min. The images were reconstructed using a three-dimensional ordered subsets expectation maximization/maximum a posteriori/ (OSEM3D/MAP) algorithm, and no correction was applied for attenuation and scatter. Statistical Analysis. The data are expressed as means ( SD. One-way analysis of variance (ANOVA) was used for statistical evaluation. A P-value of 98% based on analytical HPLC and a specific activity of 40-50 GBq/µmol. As expected, the retention time for [18F]4 (11.1 min) was significantly higher than for [18F]5 (9.75 min), reflecting the higher lipophilicity of [18F]4 compared to that of [18F]5. The stability of [18F]4 and [18F]5 was determined following incubation in mouse plasma at 37 °C over 2 h with samples collected for analysis by radio-HPLC at 30 min, 1 h, and

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Figure 1. Above chromatogram: [18 F]4 following incubation in mouse plasma after 1 h. Right peak at tR ) 11 min is intact [18 F]4. Lower chromatogram: [18 F]5 incubation in mouse plasma after 1 h. Main peak is intact [18 F]5.

2 h. Plasma studies with [18F]5 revealed no detectable degradation over 2 h. On the other hand, conjugate [18F]4 demonstrated poor stability in plasma with only 5% of the intact tracer remaining after 2 h (Figure 1). Subsequent studies of 4 in phosphate buffer at different pH values revealed that the stability was pH-dependent. At pH 7.3, 17% of 4 remained intact after 60 min, while at pH 6, 50% remained intact after 60 min. At pH 4, no degradation of 4 was observed within the time frame of the study (2 h). The observed degradation was attributed to the retro-Michael addition of the prosthetic group favored at higher pH. LCMS studies supported this assumption with reappearance of peptide precursor 2 being confirmed in a stock solution of 4 stored at pH 7 (data not shown) (20). Since [18F]4 proved to be unstable in mice plasma, log P experiments were conducted only for [18F]5. The log P (octanol/water) for [18F]5 was -2.61 ( 0.01 confirming the hydrophilic nature of the compound. The affinity of compound 5 for Rvβ3 integrin was determined in a competitive binding assay with 125I-echistatin. Binding of 125 I-echistatin to Rvβ3 was competed out by 5 in a concentrationdependent manner with a measured Ki value of 3.0 nM as shown in Figure 2. This suggests that attachment of the prosthetic group to the peptide had no effect on receptor binding, with the reported Ki for NC100717 being 6.8 nM (16). The unstable nature of 4 precluded an accurate interpretation of the Ki for this compound. Despite its poor plasma stability, dynamic microPET scans were performed with [18F]4 in three tumor-bearing mice. We rationalized that, due to low interstitial pH caused by hypoxia and metabolic activity in the lesion, any intact [18F]4 distributed to the local tumor environment would display greater stability (21-23). The more rapid degradation of background [18F]4 at normal physiological conditions would result in a more rapid excretion of the prosthetic group improving the signal-to-noise ratios. Lewis et al. have also

Figure 2. Competition of 125I-echistatin by 5 (Ki ) 3 nM) on EAHy926 cell membranes (2). Echistatin for reference (().

conducted similar studies with a labile maleimidocysteineamido-DOTA side chain (24, 25). However, this hypothesis proved to be incorrect with the rapid degradation in blood preventing tumor accumulation, and thus, [18F]4 was not evaluated further. The results of the biodistribution of [18F]5 in mice with subcutaneously implanted OHS tumors are shown in Figure 3. The tracer cleared rapidly from the blood (0.18% ID/g, 2 h postinjection) and had good tumor uptake (3.48% ID/g) 2 h postinjection, low bone uptake indicating little or no defluorination, and a favorable biodistribution profile. At 120 min post-injection, the tumor to blood and tumor to muscle ratios were 19 and 5, respectively. The systemic clearance was predominately by renal excretion. In a separate experiment addressing specificity, mice were coinjected with [18F]5 and 10 mg/kg of nonradioactive RGD peptide NC100717. The blocking study demonstrated a >10fold reduction in tumor uptake at 120 min post-injection (3.48% ID/g vs 0.25% ID/g, P < 0.05) indicating Rvβ3-mediated tumor binding. Uptake in nontarget organs was also reduced in the presence of excess unlabeled RGD peptide, indicating that the uptake in these tissues was at least partly Rvβ3-mediated;

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Figure 3. Radioactivity expressed as percentage injected dose per gram tissue in the tumor and key organs of the subcutaneous human OHS xenograft mouse model 5 and 120 min after intravenous injection of approximately 1 MBq [18F]5 at. (n ) 4 for each time point).

Figure 4. Tumour to nontarget ratios of key organs for [18F]5 without and in the presence of coinjected excess NC100717.

however, tumor uptake was reduced most markedly (Figure 4). Similar observations have been reported with other radiolabeled RGD peptides (26-29). MicroPET images (15 min static single frame) were taken at 105 min of one mouse bearing an OHS tumor injected with 5.2 MBq [18F]5 as shown in Figure 5. The tumor was clearly visible with high tumor to background activity ratio. Highest uptake of radioactivity was seen in the kidneys and to a lesser extent in the liver/GI region, which is in agreement with the data obtained from direct tissue sampling. In a similar study, the specificity of [18F]5 for the Rvβ3 integrin in vivo was confirmed by a blocking experiment where the tracer was coinjected with nonlabeled cyclic RGD peptide (10 mg/kg). As can be seen from Figure 6, uptake in OHS tumor in the presence of nonradiolabeled RGD peptide was clearly lower than without

RGD blocking, which is in good agreement with the biodistribution studies.

DISCUSSION There have been several reports on the use of various 18Fprosthetic groups for labeling of peptides for in vivo PET studies (30, 31). However, many of these strategies are dependent on acylation reactions (32-34) which, for peptide sequences containing more than one free amino group, lead to the formation of a multitude of products. Site-selective chemistries are available using, for example, click chemistry, oxime bond formation, and alkylation reactions with thiols (18Fmaleimide, 18F-thiols), which produce a single radiolabeled product (35, 36). However, all the above approaches have their limitations. The click chemistry requires a copper catalyst and

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Figure 5. Coronal (left), transaxial (upper right), and sagittal (lower right) microPET images (15 min static single frame) at 105 min postinjection of an OHS tumor-bearing mouse injected with 5.2 MBq [18F]5. K ) kidneys. Bl ) bladder. White arrow indicates tumor.

Figure 6. Coronal microPET images of OHS tumor-bearing mice at 105 min post-injection of [18F]5 without (A) and with (B) coinjection of nonradioactive RGD peptide (10 mg/kg). Tumors are indicated by white arrows.

is complicated by a distillation step which is nontrivial with respect to automation. Free sulfhydryl groups are relatively uncommon in most peptides and are furthermore susceptible to oxidation and disulfide formation. In this study, the site-selective introduction of an 18F-Nmethylaminooxy prosthetic group into an RGD peptide was explored, with the peptide component bearing two different types of Michael acceptor moiety. The 18F-FENMA-nitrostyrene-RGD ([18F]4) conjugate displayed pH-dependent degradation with poor stability in the physiological to alkaline pH range. Improved stability was observed at lower pH values with no observable degradation of the conjugate at pH 4. The hypothesis of enhanced product stability in the tumor interstitia combined with rapid clearance from the background was tested (vide supra), but no tumor uptake was observed. The reason for this was likely a rapid retro-Michael addition of the 18F-FENMA from the nitrostyrene in vivo. It remains to be explored whether substituents other than the nitro group on the styrene moiety can render sufficient stability to the conjugates. The conjugation kinetics of 18F-FENMA with the vinylsulfone peptide 3 were slower than for the nitrostyrene derivative, requiring a 60 min reaction time; however, using 5 mg of

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precursor 3 a yield of 8.5-12% (decay-corrected postformulation) of the drug product [18F]5 was obtained. In contrast to [18F]4, [18F]5 demonstrated excellent stability in mouse plasma over two hours, and was more hydrophilic as reflected by a shorter HPLC retention time. However, for clinical utility of this methodology, further optimization with respect to synthesis yield and shorter synthesis time is a prerequisite. The two cysteic acid residues in the peptide precursor contribute to the high hydrophilicity of the conjugate as the sidechains remain deprotonated at physiological pH. Biodistribution studies with [18F]5 demonstrated that the tracer was excreted mainly through the renal system reflecting its hydrophilic nature. As a further demonstration of in vivo stability, the initial low uptake and subsequent wash-out of radioactivity over time in the femurs of the mice suggested that no significant defluoridation of [18F]5 was taking place. Uptake in nontarget organs was most pronounced in kidney, followed by liver, gut, and skin. The high kidney uptake may be Rvβ3 mediated, as rodent kidneys have been found to express integrin in the endothelial cells of the small vessels in the glomerulus (37). The data presented herein are similar to those published for the 18Fgalacto-RGD studied in osteosarcoma-bearing mice with higher uptake values for organ and tumor for [18F]5. The slight differences in biodistribution may be a result of the higher log P of [18F]5 compared to that of 18F-galacto-RGD (-2.61 and -3.17, respectively), which increases the relative retention of this peptide in vivo (38). The xenograft model used in this study was an osteosarcoma model derived from human tissue. Direct immunohistochemical staining of Rvβ3-integrin expression was not possible due to the absence of a suitable antibody specific for mice (39). Staining with CD31 demonstrated vascularization of the tumors. The assumption that [18F]5 was able to target the neovasculature via the Rvβ3 receptors expressed on endothelial cells was supported by the work of Pasqualini et al. who demonstrated that tumor vessels are the structural elements predominantly targeted by the RGD sequence (40). Tracer uptake was significantly higher in nonblocked animals than those coinjected with 10 µg/g nonradioactive RGD peptide, suggesting a specificity for Rvβ3 expression in the human OHS osteosarcoma. This was in accordance with published data from other groups working with alternative osteosarcoma mouse model where the tumors were induced using 90Sr (38, 27). The relatively large standard deviations in tumor uptake observed at 2 h post-injection ((1.27% ID/g) compared to nontarget organs ((0.28% ID/g) may reflect the difference in tumor sizes among the mice (0.1-0.4 g) used in the study. Tumor size is likely to impact the degree of neovascularization and Rvβ3 expression, as new blood vessels are required to supply oxygen and nutrients when the lesion reaches a diameter of around 1-2 mm (41). Although the data set was not large enough to reach full statistical significance, plots of tumor uptake (%ID/g) of the tracer against tumor weight indicated a sufficient correlation (R ) 0.79). Similar observations have been reported previously in the literature with Rvβ3 specific radioligands (28).

CONCLUSION In conclusion, the prosthetic group O-(2-(2-[18F]fluoroethoxy)ethyl)-N-methylhydroxylamine (18F-FENMA) was found to have promising characteristics for in vivo use in combination with a vinylsulfonyl modified RGD peptide in human OHS xenograft bearing mice. In vitro studies demonstrated high integrin Rvβ3 binding affinity and good in vitro plasma stability. MicroPET showed good visualization of tumor along with good tumor-to-background ratios. The excretion of 18F[5] was predominately by the renal route.

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F-FENMA Conjugated to Cyclic RGD Peptides

ACKNOWLEDGMENT Many thanks to Prof. Erik Dissen and Ph.D. student Per Christian Sæther (Dept of Anatomy, University of Oslo) for giving us access to the gamma counter and the included excellent technical support.

LITERATURE CITED (1) Volkow, N. D., Mullani, N. A., and Bendriem, B. (1988) Positron emission tomography instrumentation: an overview. Am. J. Physiol. Imaging 3, 142–53. (2) Cai, L., Lu, S., and Pike, V. W. (2008) Chemistry with [18F]fluoride ion. Eur. J. Org. Chem. 17, 2853–73. (3) Helus, F., Maier-Borst, W., Sahm, U., and Wiebe, L. I. (1979) The fluorine-18 cyclotron production methods. Radiochem. Radioanal. Lett. 38, 395–410. (4) Guillaume, M., Luxen, A., Nebeling, B., Argentini, M., Clark, J. C., and Pike, V. W. (1991) Recommendations for fluorine-18 production. Appl. Radiat. Isot. 42, 749–62. (5) Lasne, M. C., Perrio, C., Rouden, J., Barre, L., Roeda, D., Dolle, F., and Crouzel, C. (2002) Chemistry of β+-emitting compounds based on fluorine-18. Top. Curr. Chem. 222, 201–258. (6) Okarvi, S. M. (2001) Recent progress in fluorine-18 labelled peptide radiopharmaceuticals. Eur. J. Nucl. Med. 28, 929–938. (7) Poethko, T., Schottelius, M., Thumshirn, G., Herz, M., Haubner, R., Henriksen, G., Kessler, H., Schwaiger, M., and Welter, H. J. (2004) Chemoselective pre-conjugate radiohalogenation of unprotected mono- and multimeric peptides via oxime formation. Radiochim. Acta 92, 317–327. (8) Glaser, M., and Robins, E. G. (2009) Click labelling in PET radiochemistry. J. Labelled. Compd. Radiopharm. 52, 407–414. (9) Kilbourn, M. R., Dence, C. S., Welch, M. J., and Mathias, C. J. (1987) Fluorine-18 labeling of proteins. J. Nucl. Med. 28, 462– 470. (10) Bruus-Jensen, K., Poethko, T., Schottelius, M., Hauser, A., Schwaiger, M., and Wester, H. J. (2006) Chemoselective hydrazone formation between HYNIC-functionalized peptides and 18F-fluorinated aldehydes. Nucl. Med. Biol. 33, 173–183. (11) Glaser, M., Morrison, M., Solbakken, M., Arukwe, J., Karlsen, H., Wiggen, U., Champion, S., Kindberg, G. M., and Cuthberstson, A. (2008) Radiosynthesis and biodistribution of cyclic RGD peptides conjugated with novel [18F]fluorinated aldehydecontaining prosthetic groups. Bioconjugate Chem. 19, 951–957. (12) Wu, Z., Li, Z. B., Cai, W., He, L., Chin, F. T., Li, F., and Chen, X. (2007) 18F-labeled mini-PEG spacered RGD dimer (18F-FPRGD2): synthesis and microPET imaging of Rvβ3 integrin expression. Eur. J. Nucl. Med. Mol. Imaging 34, 1823– 1831. (13) Wu, Z., Li, Z. B., Chen, K., Cai, W., He, L., Chin, F. T., Li, F., and Chen, X. (2007) MicroPET of tumor integrin Rvβ3 expression using 18F-labeled PEGylated tetrameric RGD peptide (18F-FPRGD4). J. Nucl. Med. 48, 1536–1544. (14) Olberg, D. E., Hjelstuen, O. K., Solbakken, M., Arukwe, J., Karlsen, H., and Cuthbertson, A. (2008) A novel prosthetic group for site-selective labeling of peptides for positron emission tomography. Bioconjugate Chem. 19, 1301–1308. (15) Olberg, D. E., Hjelstuen, O. K., Solbakken, M., Arukwe, J. M., Dyrstad, K., and Cuthbertson, A. (2009) Site-specific addition of an 18F-N-methylaminooxy-containing prosthetic group to a vinylsulfone modified peptide. J. Labelled Compd. Radiopharm. 52, 571–575. (16) Indrevoll, B., Kindberg, G. M., Solbakken, M., Bjurgert, E., Johansen, J. H., Karlsen, H., Mendizabal, M., and Cuthbertson, A. (2006) NC-100717: A versatile RGD peptide scaffold for angiogenesis imaging. Bioorg. Med. Chem. Lett. 16, 6190–6193. (17) Jia, B., Liu, Z., Shi, J., Yu, Z., Yang, Z., Zhao, H., He, Z., Liu, S., and Wang, F. (2008) Linker effects on biological properties of 111In-labeled DTPA conjugates of a cyclic RGDfK dimer. Bioconjugate Chem. 19, 201–210.

Bioconjugate Chem., Vol. 21, No. 12, 2010 2303 (18) Park, J., and Pei, D. (2004) Trans-β-nitrostyrene derivatives as slow-binding inhibitors of protein tyrosine phosphatases. Biochemistry 43, 15014–1521. (19) Fodstad, O., Brogger, A., Bruland, O., Solheim, O. P., Nesland, J. M., and Pihl, A. (1986) Characteristics of a cell line established from a patient with multiple osteosarcoma, appearing 13 years after treatment for bilateral retinoblastoma. Int. J. Cancer 38, 33–40. (20) Bernasconi, C. F., Renfrow, R. A., and Tia, P. R. (1986) Nucleophilic addition to olefins. 16. Unusual substituent effects in the reaction of amines with beta-nitrostyrenes. Solvent effect on intrinsic rate constants. J. Am. Chem. Soc. 108, 4541–4549. (21) Helmlinger, G., Yuan, F., Dellian, M., and Jain, R. K. (1997) Interstitial pH and pO2 gradients in solid tumors in vivo: Highresolution measurements reveal a lack of correlation. Nat. Med. 3, 177–182. (22) Stubbs, M., McSheehy, P. M. J., and Griffiths, J. R. (1999) Causes and consequences of acidic pH in tumors: a magnetic resonance study. AdV. Enzyme Regul. 39, 13–30. (23) Horiuchi, K., Saji, H., and Yokoyama, A. (1998) Tc(V)-DMS tumor localization mechanism: a pH-sensitive Tc(V)-DMSenhanced target/nontarget ratio by glucose-mediated acidosis Preparation and labeling condition studies. J. Nucl. Biol. Med. 25, 549–555. (24) Lewis, M. R., and Shively, J. E. (1998) Maleimidocysteineamido-DOTA derivatives: New reagents for radiometal chelate conjugation to antibody sulfhydryl groups undergo pH-dependent cleavage reactions. Bioconjugate Chem. 9, 72–86. (25) Williams, L. E., Lewis, M. R., Bebb, G. G., Clarke, K. G., Odom-Maryon, T. L., Shively, J. E., and Raubitschek, A. A. (1998) Biodistribution of 111In- and 90Y-Labeled DOTA and maleimidocysteineamido-DOTA conjugated to chimeric anticarcinoembryonic antigen antibody in xenograft-bearing nude mice: Comparison of stable and chemically labile linker systems. Bioconjugate Chem. 9, 87–93. (26) Dijkgraaf, I., Beer, A. J., and Wester, H. J. (2009) Application of RGD-containing peptides as imaging probes for alphavbeta3 expression. Front. Biosci. 14, 887–899. (27) Haubner, R., Wester, H. J., Reuning, U., SenekowitschSchmidtke, R., Diefenbach, B., Kessler, H., Sto¨cklin, G., and Schwaiger, M. (1999) Radiolabeled Rvβ3 integrin antagonists: a new class of tracers for tumor targeting. J. Nucl. Med. 40, 1061– 1071. (28) Shi, J., Wang, L., Kim, Y. S., Zhai, S., Jia, B., Wang, F., and Liu, S. (2009) 99mTcO(MAG2-3G3-dimer): a new integrin Rvβ3-targeted SPECT radiotracer with high tumor uptake and favorable pharmacokinetics. Eur. J. Nucl. Med. Mol. Imaging 36, 1874–1884. (29) Dijkgraaf, I., Kruijtzer, J. A., Liu, S., Soede, A. C., Oyen, W. J., Corstens, F. H., Liskamp, R. M., and Boerman, O. C. (2007) Improved targeting of the Rvβ3 integrin by multimerisation of RGD peptides. Eur. J. Nucl. Med. Mol. Imaging 34, 267– 273. (30) Li, Z. B., Wu, Z., Chen, K., Chin, F. T., and Chen, X. (2009) Click chemistry for 18F-labeling of RGD peptides and microPET imaging of tumor integrin Rvβ3 expression. Bioconjugate Chem. 18, 1987–1994. (31) Liu, Z., Liu, S., Wang, F., Liu, S., and Chen, X. (2009) Noninvasive imaging of tumor integrin expression using 18Flabeled RGD dimer peptide with PEG4 linkers. Eur. J. Nucl. Med. Mol. Imaging 36, 1296–1307. (32) Wust, F., Hultsch, C., Bergmann, R., Johannsen, B., and Henle, T. (2003) Radiolabelling of isopeptide Nε-(γ-glutamyl)-L-lysine by conjugation with N-succinimidyl-4-[18F]fluorobenzoate. Appl. Radiat. Isot. 59, 43–48. (33) Liu, Z., Liu, S., Wang, F., Liu, S., and Chen, X. (2009) Noninvasive imaging of tumor integrin expression using 18Flabeled RGD dimer peptide with PEG4 linkers. Eur. J. Nucl. Med. Mol. Imaging 36, 1296–1307. (34) Wester, H. J., and Schottelius, M. (2007) Fluorine-18 labeling of peptides and proteins. PET Chemistry - The driVing force in

2304 Bioconjugate Chem., Vol. 21, No. 12, 2010 molecular imaging (Schubiger, A. P., Lehmann, L., and Friebe, M., Eds.) pp 80-104, Chapter 4, Springer, Berlin. (35) Glaser, M., Solbakken, M., Turton, D. R., Pettitt, R., Barnett, J., Arukwe, J., Karlsen, H., Cuthbertson, A., Luthra, S. K., and Arstad, E. (2009) Methods for 18F-labeling of RGD peptides: comparison of aminooxy [18F]fluorobenzaldehyde condensation with ‘click labeling’ using 2-[18F]fluoroethylazide, and Salkylation with [18F]fluoropropanethiol. Amino Acids 37, 717– 724. (36) Cai, W., Zhang, X., Wu, Y., and Chen, Z. (2006) A thiolreactive 18F-labeling agent, N-[2(4-18F-fluorobenzamido)ethyl]maleimide, and synthesis of RGD peptide-based tracer for PET imaging of Rvβ3 integrin expression. J. Nucl. Med. 47, 1172– 1180. (37) Li, Z. B., Chen, K., and Chen, X. (2008) 68Ga-labeled multimeric RGD peptides for microPET imaging of integrin Rvβ3 expression. Eur. J. Nucl. Med. Mol. Imaging 35, 1100–1108.

Olberg et al. (38) Haubner, R., Westerm, H. J., Weber, W. A., Mang, C., Ziegler, S. I., Goodman, S. L., Senekowitsch-Schmidtke, R., Kessler, H., and Schwaiger, M. (2001) Noninvasive imaging of Rvβ3 integrin expression using18F-labeled RGD-containing glycopeptide and positron emission tomography. Cancer Res. 61, 1781–1785. (39) Mulder, W., Castermans, K., van Beijnum, J., oude Egbrink, M. G., Chin, P. T., Fayad, Z. A., Lo¨wik, C. W., Kaijzel, E. L., Que, I., Storm, G., Strijkers, G. J., Griffioen, A. W., and Nicolay, K. (2009) Molecular imaging of tumor angiogenesis using Rvβ3integrin targeted multimodal quantum dots. Angiogenesis 12, 17– 24. (40) Pasqualini, R., Koivunen, E., and Ruoslahti, E. (1997) Rv integrins as receptors for tumor targeting by circulating ligands. Nat. Biotechnol. 15, 542–546. (41) Folkman, J. (1971) Tumor angiogenesis: therapeutic implications. N. Engl. J. Med. 285, 1182–1186. BC1003229