Nuclear Medicine and Biology - Articles in Press

177Lu labeling of Herceptin and preclinical validation as a new radiopharmaceutical for radioimmunotherapy of breast cancer - Corrected Proof

Samira Rasaneh, Hossein Rajabi, Mohammad Hossein Babaei, Fariba Johari Daha — 2010-09-02

Abstract: Introduction: In the present study, Herceptin was labeled with lutetium-177 via DOTA, and the necessary preclinical quality control tests (in vitro and in vivo) were performed to evaluate its use as a radioimmunotherapy agent.Material and Methods: Herceptin was conjugated to DOTA as a chelator in three different conjugation buffers (ammonium acetate, carbonate and HEPES buffer); each of the resulting conjugates was compared with respect to in vitro characteristics such as number of chelates per antibody, incorporated activity, immunoreactivity and in vitro stability in PBS buffer and blood serum. The biodistribution study and gamma camera imaging were performed in mice bearing breast tumors. To assess the therapeutic effects of 177Lu-Herceptin, cytotoxicity was investigated for 7 days in a SKBr3 breast cancer cell line.Results: Carbonate buffer was the best conjugation buffer (number of chelates per antibody: 6; incorporated activity: 81%; immunoreactivity: 87%; buffer stability: 86%; serum stability: 81%, after 4 days). The efficient tumor uptake observed in the biodistribution studies was consistent with the gamma camera image results. At a concentration of 4 μg ml−1, 177Lu-Herceptin (surviving cells: 5±0.6% of the total cells) of the total cells corresponded to an approximately eightfold increase in cytotoxicity in comparison to unmodified Herceptin (surviving cells: 43±3.9%).Conclusion: The new complex described herein could be considered for further evaluation in animals and potentially in humans as a radiopharmaceutical for use in the radioimmunotherapy of breast cancer. These results may be important for patients who cannot tolerate the therapeutic dosage of Herceptin currently used because of heart problems.

Myocardial 99mTc-sestamibi extraction and washout in hypertensive heart failure using an isolated rat heart - Corrected Proof

2010-09-02

Abstract: Purpose: Myocardial mitochondria are the primary part of energy production for healthy cardiac contraction. And mitochondrial dysfunction would play an important role in progressive heart failure. In the recent years, myocardial washout of 99mTc-sestamibi [(99mTc-hexakis-2-methoxy-2-methylpropyl isonitrile (MIBI)] has been introduced to be a potential marker in patients with heart failure. The objective of this study was to clarify MIBI extraction and washout kinetics using isolated perfusion system in hypertension induced model of myocardial dysfunction.Methods: Six-week-old Dahl-salt sensitive rats, allotted to 4 groups; a 5-week high-salt group (5wk-HS), 12-week high-salt group (12wk-HS) and two age-matched, low-salt diet control groups (5wk-LS and 12wk-LS). The rats in 5wk-HS and 12wk-HS groups were fed a high-salt diet (containing 8% NaCl). Cardiac function was examined by echocardiography before removing heart. Hearts were perfused according to the Langendorff method at a constant flow rate, in which 20-min MIBI washin was conducted followed by 25-min MIBI washout. Whole heart radioactivity was collected every sec by an external gamma detector. The myocardial extraction, K1 (ml/min) and washout rate, k2 (min−1) were generated.Results: High-salt diet groups showed significant high-blood pressure. Echocardiography revealed thickened LV walls in 5wk-HS, and reduced cardiac function in 12wk-HS, compared to each age-matched control group. K1 showed no significant difference among all groups (5wk-HS: 2.36±1.07, 5wk-control: 2.59±0.28, 12wk-HS: 1.91±0.90, and 12wk-control: 2.84±0.57). k2 in 5wk-HS was comparable to that in the age matched control group (0.00030±0.00039 vs −0.000010±0.00044), but it was increased remarkably in 18wk-HS compared to the age matched control group (0.0025±0.0011 vs 0.000025±0.000041, P<.01), and 5wk-HS (P<.01).Conclusion: In the course of hypertensive heart disease, MIBI washout was increased in the transitional state from hypertrophied to dilated and failing heart, while MIBI extraction remained intact.

A kit to prepare 111In-DTPA-trastuzumab (Herceptin) Fab fragments injection under GMP conditions for imaging or radioimmunoguided surgery of HER2-positive breast cancer - Corrected Proof

Deborah A. Scollard, Conrad Chan, Claire M.B. Holloway, Raymond M. Reilly — 2010-09-02

Abstract: Introduction: The human epidermal growth factor receptor-2 (HER2) gene is amplified in 25% of invasive breast cancers, and receptor overexpression has been noted in up to 60% of early stages of the disease [ductal carcinoma in situ (DCIS)]. Preclinical studies have revealed high tumor/blood ratios (>27:1) for 111In-labeled Fab fragments of the HER2 monoclonal antibody, trastuzumab (Herceptin) (111In-DTPA-trastuzumab Fab) at 72 h pi in athymic mice bearing subcutaneous human breast cancer xenografts. Our aim in this study was to formulate a kit for preparation of 111In-DTPA-trastuzumab Fab injection under good manufacturing practice (GMP) conditions suitable for human administration in a Phase I clinical trial of imaging and radioimmunoguided surgery (RIGS) of HER2-positive breast cancer.Methods: Fab fragments were produced by digestion of trastuzumab IgG (Herceptin) with immobilized papain for 20 h at 37°C. Fab fragments were purified by ultrafiltration, then reacted with a 10-fold molar excess of diethylenetriaminepentaacetic acid (DTPA) dianhydride. DTPA-Fab fragments were purified, then sterilized by filtration into unit dose glass vials (kits). Kits were tested against specifications for volume (0.9–1.1 ml), protein concentration (0.45–0.55 mg/ml), pH (5.5–6.5), DTPA substitution (0.5–4.0 mol DTPA/mol Fab), appearance (clear, colorless and particle free), labeling efficiency (≥85%), and sterility and apyrogenicity (USP XXXII). Immunoreactivity of 111In-DTPA-trastuzumab Fab towards HER2 was measured by saturation radioligand binding assays using SKBR-3 human breast cancer cells (specifications: Ka=0.6–9.6×107 L/mol; Bmax=0.6–10.4×106 sites/cell). 111In-DTPA-trastuzumab Fab injection was prepared by adding 80–100 MBq of 111InCl3 to a single kit vial and incubating for 30 min at room temperature. 111In-DTPA-trastuzumab Fab was assayed for the amount of radioactivity and tested for pH, radiochemical purity (RCP), appearance and sterility.Results: Pure and homogeneous Fab fragments were produced. Eleven lots of kits met established quality specifications. The labeling efficiency with 111In was 90.6±2.2%. 111In-DTPA-trastuzumab Fab bound specifically to HER2 on SKBR-3 cells (Ka=4.8±2.5×107 L/mol and Bmax=1.6±0.8×106 sites/cell). Thirteen lots of 111In-DTPA-trastuzumab injection met all established specifications. Kits were stable for 90 days and 111In-DTPA-trastuzumab Fab injection was stable for 24 h stored at 4°C.Conclusions: A kit was formulated under GMP conditions for the preparation of 111In-DTPA-trastuzumab Fab injection suitable for human administration. The kits were approved by Health Canada.

Neurotensin(8–13) analogue: radiolabeling and biological evaluation using different chelators - Corrected Proof

2010-09-02

Abstract: Introduction: Several strategies on the development of radiopharmaceuticals have been employed. Bifunctional chelators seem to be a promising approach since high radiochemical yields as well as good in vitro and in vivo stability have been achieved. To date, neurotensin analogs have been radiolabeled using the 99mTc-carbonyl approach and none was described employing the bifunctional chelating agent technique.Aim: The purpose of this study was to evaluate the radiochemical and biological behaviour of NT(8–13) analogue radiolabeled with 99mTc, using HYNIC and NHS-S-acetyl-MAG3 as chelator agents.Methods: Radiolabeling, in vitro stability toward cysteine and glutathione, partition coefficient and plasma protein binding were assessed for both radioconjugates. Biodistribution in healthy Swiss mice were carried out in order to evaluate the biological behaviour of the radiocomplexes.Results: Radiochemical yields were higher than 97% and no apparent instability toward transchelant agents was observed for both radioconjugates. A higher lipophilic character was observed for the radioconjugate labeled via MAG3. The chelators seem to have no effect on the percentage of the radioconjugate bound to plasma proteins. A similar biological pattern was observed for both radioconjugates. Total blood, bone and muscle values revealed a slightly slower clearance for the radiocomplex labeled via MAG3. Moreover, a remarkable liver and intestinal uptake was observed for the radiocomplex labeled via MAG3 even at the later time points studied.Conclusion: The high radiochemical yields achieved and the similar in vivo pattern found for both radioconjugates make them potential candidates for imaging tumors using nuclear medicine techniques.

Radiosynthesis and pre-clinical evaluation of [18F]fluoro-[1,2-2H4]choline - Corrected Proof

2010-09-02

Abstract: Introduction: Choline radiotracers are widely used for clinical PET diagnosis in oncology. [11C]Choline finds particular utility in the imaging of brain and prostate tumor metabolic status, where 2-[18F]fluoro-2-deoxy-d-glucose (‘FDG’) shows high background uptake. More recently we have extended the clinical utility of [11C]choline to breast cancer where radiotracer uptake correlates with tumor aggressiveness (grade). In the present study, a new choline analog, [18F]fluoro-[1,2-2H4]choline, was synthesized and evaluated as a potential PET imaging probe.Methods: [18F]Fluorocholine, [18F]fluoro-[1-2H2]choline and [18F]fluoro-[1,2-2H4]choline were synthesized by alkylation of the relevant precursor with [18F]fluorobromomethane or [18F]fluoromethyl tosylate. Radiosynthesis of [18F]fluoromethyl tosylate required extensive modification of the existing method. [18F]Fluorocholine and [18F]fluoro-[1,2-2H4]choline were then subjected to in vitro oxidative stability analysis in a chemical oxidation model using potassium permanganate and an enzymatic model using choline oxidase. The two radiotracers, together with the corresponding di-deuterated compound, [18F]fluoro-[1-2H2]choline, were then evaluated in vivo in a time-course biodistribution study in HCT-116 tumor-bearing mice.Results: Alkylation with [18F]fluoromethyl tosylate proved to be the most reliable radiosynthetic route. Stability models indicate that [18F]fluoro-[1,2-2H4]choline possesses increased chemical and enzymatic (choline oxidase) oxidative stability relative to [18F]fluorocholine. The distribution of the three radiotracers, [18F]fluorocholine, [18F]fluoro-[1-2H2]choline and [18F]fluoro-[1,2-2H4]choline, showed a similar uptake profile in most organs. Crucially, tumor uptake of [18F]fluoro-[1,2-2H4]choline was significantly increased at late time points compared to [18F]fluorocholine and [18F]fluoro-[1-2H2]choline.Conclusions: Stability analysis and biodistribution suggest that [18F]fluoro-[1,2-2H4]choline warrants further in vivo investigation as a PET probe of choline metabolism.

Tetradentate bis-phosphine ligands (P2N2 and P2S2) and their Rh(III), Ni(II) and 105Rh Complexes: X-ray crystal structures of trans-[RhCl2(L2)]PF6, [Ni(L2)](PF6)2 and μ-O2SO2-[Ni(L5)]2(PF6)2 - Corrected Proof

2010-09-02

Abstract: Introduction: Tetradentate acyclic and macrocyclic diphosphine ligands (P2N2 and P2S2) have been synthesized and characterized as potential chelates for Rh(III).Methods: The coordination complexes [RhCl2(L1)]Cl, trans-[RhCl2(L2)]PF6, [Ni(L2)](PF6)2, [Ni(L3)](PF6)2, [RhCl2(L4)]PF6 and [RhCl2(L5)]PF6 have been synthesized and characterized. In addition, radiochemistry studies of the 105Rh complexes with the ligands N,N′-bis[2-(diphenylphosphino)phenyl]-1,3-diaminopropane (L1), 4,8-diphenyl-1,11-diaza-4,8-diphosphaundecane (L2), 5,9-diphenyl-5,9-diphospha-2,12-dithiatridecane (L3) and 1,4,8,11-tetraphenyl-4,8-diphospha-1,11-dithiaundecane (L4) are reported, including normal mouse biodistributions of 105Rh–L2.Results: trans-[RhCl2(L2)]PF6 crystallized in the monoclinic space group P21/c with a=9.9353(5) Å, b=9.0929(5) Å, c=28.689(1) Å, β=93.1400(10) deg, Z=4, R=0.037 and Rw=0.053. [Ni(L2)](PF6)2 crystallized in the monoclinic space group P21/c with a=11.9665(6) Å, b=14.8903(7) Å, c=31.148(1) Å, β=91.587(1) deg, Z=8, R=0.056 and Rw=0.083. μ-O2SO2-[Ni(L5)]2(PF6)2, an unusual sulfate-bridged Ni(II) dimer, crystallized in the triclinic space group P1 bar with a=15.179(2) Å, b=15.514(2) Å, c=16.128(2) Å, α=105.280(7) deg, β=113.074(6) deg, γ=101.657(8) deg, Z=2, R=0.050 and Rw=0.072.Conclusions: Phosphine-containing ligands allowed for lower temperatures and lower ethanol concentrations in the formulations for 105Rh(L) complexation, but at the expense of higher ligand concentrations.

The stability of methyl-, ethyl- and fluoroethylesters against carboxylesterases in vitro: there is no difference - Corrected Proof

2010-09-02

Abstract: Introduction: Carboxylesterases (CES) play a very important role in the hydrophilic biotransformation of a huge number of structurally diverse drugs and especially play a leading part in the catabolic pathway of carboxylesters or thioesters. Hence, the aim of the present study was the comparison of the in vitro stability of methyl- and ethylesters with fluoroethylesters.Methods: We incubated methyl 3β-(4-iodophenyl)tropane-2β-carboxylate (β-CIT)/2-fluoroethyl 3β-(4-iodophenyl)tropane-2β-carboxylate (FE@CIT), methyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate (MTO)/ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate (ETO)/2-fluoroethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate (FETO), ethyl 8-fluoro-5-methyl-6-oxo-5,6-dihydro-4H-benzo-[f]imidazo[1,5-a]-[1,4]diazepine-3-carboxylate (FMZ)/2-fluoroethyl 8-fluoro-5-methyl-6-oxo-5,6-dihydro-4H-benzo-[f]imidazo[1,5-a]-[1,4]diazepine-3-carboxylate (FFMZ), methyl 1-phenylethyl-4-(N-propanoylanilino)piperidine-4-carboxylate (CFN)/2-fluoroethyl 1-phenylethyl-4-(N-propanoylanilino)piperidine-4-carboxylate (FE@CFN) and methyl 2,4-diethyl-3-methylsulfanylcarbonyl-6-phenylpyridine-5-carboxylate [(Me)2@SUPPY]/2-fluorethyl 2,4-diethyl-3-ethylsulfanylcarbonyl-6-phenylpyridine-5-carboxylate (FE@SUPPY) under physiological conditions. The enzymatic reactions were stopped at different time points and analyzed by a standard protocol.Results: The Michaelis–Menten constants (KM) and limiting velocities (Vmax) are comparable. The statistical KM values were as follows: β-CIT/FE@CIT, P>.05; MTO/FETO, P>.06; ETO/FETO, P>.09; FMZ/FFMZ, P>.05; CFN/FE@CFN, P>.9; (Me)2@SUPPY/FE@SUPPY, P>.07.Conclusion: We found no statistical difference in stability against CES in vitro. These findings support the strategy to translate C-11-methyl-/ethylesters into their longer-lived F-18-fluoroethyl analogues.

Production of large quantities of 90Y by ion-exchange chromatography using an organic resin and a chelating agent - Corrected Proof

2010-08-18

Abstract: The performance of a system composed of an organic cation exchanger (Dowex 50Wx8) and a chelating agent (EDTA) previously described for the successful production of 90Y via a 90Sr/90Y generator is assessed under dynamic conditions. In an attempt to overcome the established limitation of ion-exchange resins for the separation of subcurie quantities of activity, 90Y is repeatedly isolated from an 11.8-GBq (320 mCi) 90Sr cow using a three-column tandem arrangement. The high recovery and radionuclidic purity obtained for 90Y and the parameters of the separation (time, eluant concentration, pH and flow rate range) strongly suggest that Ci quantities of 90Y can be handled satisfactorily by the ion-exchange method. No replacement or treatment of the cow, low waste generation and 90Sr losses less than 0.1% after each run were observed during the present study which, in combination with the low cost of this resin, may result in an attractive alternate method for the production of large quantities of 90Y.

Facile and rapid one-step radiosynthesis of [18F]BAY94-9172 with a new precursor - Corrected Proof

Hongliang Wang, Huixian Shi, Haiyan Yu, Shende Jiang, Ganghua Tang — 2010-08-18

Abstract: Introduction: [18F]BAY94-9172 (Florbetaben) (Compound 8) is a positron emission tomography (PET) tracer that is currently in Phase III study for in vivo mapping of fibrillar amyloid β as a pathological hallmark for Alzheimer's disease. This work reports new methods for the synthesis of [19F]BAY94-9172 and its two different precursors and radiosynthesis of [18F]BAY94-9172 with the two precursors by purification using Sep-Pak cartridge.Methods: The reference standard [19F]BAY94-9172 and the new precursor (Compound 9) were obtained from the reactions of (E)-4-methylamino-4′-hydroxystilbene (Compound 1) with methanesulfonic acid 2-[2-(2-fluoro-ethoxy)-ethoxy]-ethyl ester (Compound 11) and methanesulfonic acid 2-[2-(2-methanesulfonyloxy-ethoxy)-ethoxy]-ethyl ester (Compound 13), respectively. The reported precursor (Compound 6) is an N-BOC-protected mesylate compound, which was obtained from Compound 9. The one-step radiosynthesis of [18F]BAY94-9172 was carried out in the modified PET-MF-2V-IT-1 synthesizer by [18F]fluorination of the new precursor (Compound 9) and purification with plus C18 Sep-Pak cartridges and was compared with two-step one-pot radiosynthesis using the reported precursor (Compound 6) and Sep-Pak cartridge purification.Results: For one-step radiosynthesis, the uncorrected radiochemical yield of [18F]BAY94-9172 was 23±3% (n=5, based on [18F]fluoride) within 30 min and the radiochemical purity was greater than 95%. For two-step one-pot radiosynthesis, the uncorrected radiochemical yield of [18F]BAY94-9172 was 17±2% in 45 min (n=4, based on [18F]fluoride) with the radiochemical purity being above 95% after the Sep-Pak cartridge purification.Conclusion: [19F]BAY94-9172 and the two precursors were synthesized by a short synthetic route. Compared with HPLC purification, the use of Sep-Pak purification of [18F]BAY94-9172 reduced the total radiosynthesis time. The one-step radiosynthesis of [18F]BAY94-9172 is convenient and can easily be applied to the commercial PET tracer synthesizer for automated synthesis.

Effect of cetuximab in combination with alpha-radioimmunotherapy in cultured squamous cell carcinomas - Corrected Proof

Marika Nestor, Magnus Sundström, Matti Anniko, Vladimir Tolmachev — 2010-08-18

Abstract: Aim: The monoclonal antibody cetuximab, targeting the epidermal growth factor receptor (EGFR), is a promising molecular targeting agent to be used in combination with radiation for anticancer therapy. In this study, effects of cetuximab in combination with alpha-emitting radioimmunotherapy (RIT) in a panel of cultured human squamous cell carcinomas (SCCs) were assessed.Methods: SCC cell lines were characterized and treated with cetuximab in combination with anti-CD44v6 RIT using the astatinated chimeric monoclonal antibody U36 (211At-cMAb U36). Effects on 211At-cMAb U36 uptake, internalization and cell proliferation were then assessed in SCC cells.Results: Cetuximab in combination with 211At-cMAb U36 mediated increased growth inhibition compared to RIT or cetuximab alone in two cell lines. However, cetuximab also mediated radioprotective effects compared to RIT alone in two cell lines. The radioprotective effects occurred in the cell lines in which cetuximab clearly inhibited cell growth during radiation exposure. Cetuximab treatment also influenced 211At-cMAb-U36 uptake and internalization, suggesting interactions between CD44v6 and EGFR.Conclusions: Results from this study demonstrate the vast importance of further clarifying the mechanisms of cetuximab and radiation response, and the relationship between EGFR and suitable RIT targets. This is important not only in order to avoid potential radioprotective effects, but also in order to find and utilize potential synergistic effects from these combinations.

Comparison of the stability of Y-90-, Lu-177- and Ga-68- labeled human serum albumin microspheres (DOTA-HSAM) - Corrected Proof

Gerd Wunderlich, Eik Schiller, Ralf Bergmann, Hans-Jürgen Pietzsch — 2010-07-26

Abstract: Introduction: Microparticles derived from denatured human serum albumin (DOTA-derivatized human serum albumin microspheres, or DOTA-HSAM) are attractive carriers of radionuclides for both therapeutic and diagnostic purposes. In this article, we describe a labeling procedure for diagnostic (Ga-68) and therapeutic (Y-90, Lu-177) radionuclides and report on the results of stability studies of these products.Methods: DOTA-HSAM was labeled in 0.5 M ammonium acetate buffer, pH 5.0, containing 0.02 mg/ml detergent. After adding the radionuclide, the mixture was shaken for 15 min at 90°C. Labeling yields and in vitro stability were determined by thin-layer chromatography. For determination of the in vivo stability of Ga-68 and Y-90 DOTA-HSAM, the particles were injected intravenously in Wistar rats.Results: Labeling yields up to 95% in the case of Ga-68 and Lu-177 were achieved. Ga-68-labeled DOTA-HSAM showed high in vitro and in vivo stability. The amount of particle-bound radioactivity of Lu-177 DOTA-HSAM declines slowly in a linear manner to approximately 72% after 13 days. For Y-90, the labeling yield decreased with increasing radioactivity level. We presume radiolysis as the reason for these findings.Conclusion: The labeling of DOTA-HSAM with different radionuclides is easy to perform. The radiation-induced cleavage of the labeled chelator together with the rather short half-life of radioactivity fixation in vivo (3.7 days) is, in our opinion, opposed to therapeutic applications of DOTA-HSAM. On the other hand, the high stability of Ga-68 DOTA-HSAM makes them an attractive candidate for the measurement of regional perfusion by PET.

Technetium-99m-labeled Arg-Gly-Asp-conjugated alpha-melanocyte stimulating hormone hybrid peptides for human melanoma imaging - Corrected Proof

Jianquan Yang, Haixun Guo, Yubin Miao — 2010-07-26

Abstract: Introduction: The purpose of this study was to examine whether 99mTc-labeled Arg-Gly-Asp (RGD)-conjugated alpha-melanocyte stimulating hormone (α-MSH) hybrid peptide targeting both melanocortin-1 (MC1) and αvβ3 integrin receptors was superior in melanoma targeting to 99mTc-labeled α-MSH or RGD peptide targeting only the MC1 or αvβ3 integrin receptor.Methods: RGD-Lys-(Arg11)CCMSH, RAD-Lys-(Arg11)CCMSH and RGD-Lys-(Arg11)CCMSHscramble were designed to target both MC1 and αvβ3 integrin receptors, MC1 receptor only and αvβ3 integrin receptor only, respectively. The MC1 or αvβ3 integrin receptor binding affinities of three peptides were determined in M21 human melanoma cells. The melanoma targeting properties of 99mTc-labeled RGD-Lys-(Arg11)CCMSH, RAD-Lys-(Arg11)CCMSH and RGD-Lys-(Arg11)CCMSHscramble were determined in M21 human melanoma-xenografted nude mice. Meanwhile, the melanoma uptake of 99mTc-RGD-Lys-(Arg11)CCMSH was blocked with various non-radiolabeled peptides in M21 melanoma xenografts.Results: RGD-Lys-(Arg11)CCMSH displayed 2.0 and 403 nM binding affinities to both MC1 and αvβ3 integrin receptors, whereas RAD-Lys-(Arg11)CCMSH or RGD-Lys-(Arg11)CCMSHscramble lost their αvβ3 integrin receptor binding affinity by greater than 248-fold or MC1 receptor binding affinity by more than 100-fold, respectively. The melanoma uptake of 99mTc-RGD-Lys-(Arg11)CCMSH was 2.49 and 2.24 times (P < .05) the melanoma uptakes of 99mTc-RAD-Lys-(Arg11)CCMSH and 99mTc-RGD-Lys-(Arg11)CCMSHscramble at 2 h post-injection, respectively. Either RGD or (Arg11)CCMSH peptide co-injection could block 42% and 57% of the tumor uptake of 99mTc-RGD-Lys-(Arg11)CCMSH, whereas the coinjection of RGD+(Arg11)CCMSH peptide mixture could block 66% of the tumor uptake of 99mTc-RGD-Lys-(Arg11)CCMSH.Conclusions: Targeting both MC1 and αvβ3 integrin receptors enhanced the melanoma uptake of 99mTc-RGD-Lys-(Arg11)CCMSH in M21 human melanoma xenografts. Flank M21 human melanoma tumors were clearly visualized by single photon emission computed tomography/computed tomographic imaging using 99mTc-RGD-Lys-(Arg11)CCMSH as an imaging probe, highlighting its potential use as a dual-receptor-targeting imaging probe for human melanoma detection.

Rat pancreas uptake of [11C]dihydrotetrabenazine stereoisomers - Corrected Proof

Michael R. Kilbourn — 2010-07-26

Abstract: (+)-α-[11C]Dihydrotetrabenazine ((+)-[11C]DTBZ), a radioligand for the vesicular monoamine transporter type 2 (VMAT2), has been previously proposed as an in vivo marker of beta-cell degeneration in the pancreas. The stereospecificity of uptake of [11C]DTBZ into rat pancreas was examined here using radiolabeled forms of the (+)- and (−)-isomers. Pancreas localization of (+)-[11C]DTBZ could be partially blocked by prior administration of unlabeled (+)-DTBZ. Pancreatic uptake of the (−)-isomer was unexpectedly high and could not be blocked by pretreatment with (+)-DTBZ, but could be significantly reduced by treatment with racemic tetrabenazine, an in vivo source of (−)-DTBZ. These studies indicate that the inactive isomer of DTBZ does not provide a mechanism for defining the nonspecific binding of (+)-DTBZ in rat pancreas.

Treatment of transplanted tumor of lung adenocarcinoma A549 transfected by human somatostatin receptor subtype 2 (hsstr2) gene with 188Re-RC-160 - Corrected Proof

Rong Zhao, Weidong Yang, Zhe Wang, Guoquan Li, Weiwei Qin, Jing Wang — 2010-07-26

Abstract: Background and aim: Radionuclide-labeled somatostatin analogues selectively target somatostatin receptor (SSTR)-expressing tumors as a basis for diagnosis and treatment of these tumors. To those tumors without somatostatin receptor expressed, the hSSTR2 gene was transfected. Express of the hSSTR2 receptor was imaging and the radiotherapeutic effect was evaluated with 188Re-RC-160.Methods: The stable hSSTR2-expressing A549 cells (pcDNA3-hSSTR2 A549) and non-somatostatin receptor expressing A549 cells (pcDNA3 A549) were selected by western blot. Later, a corresponding animal tumor model was established. Expression of the hSSTR2 reporter was imaged using 188Re-RC-160 recognition. Tumors were evaluated for somatostatin receptor expression using immunohistochemistry. The distribution of 188Re-RC-160 in the animal tumor model was measured and the inhibitory effects of 188Re-RC-160 were evaluated by measurement of tumor growth and hematoxylin and eosin and TdT mediated dUTP nick end labeling (TUNEL) staining.Results: In vivo radioimaging revealed specific targeting of 188Re-RC-160 to tumors derived from pcDNA3- hSSTR2 A549 cells, compared to those from pcDNA3 A549 cells. pcDNA3- hSSTR2 A549 tumor growth inhibition was significantly higher in the single 7.4 MBq 188Re-RC-160 treatment group than in the 2×7.4 MBq rhenium-188, RC-160 group, control group, and pcDNA3 A549 tumors (P<.05). Furthermore, treatment fractionation group (2×7.4 MBq 188Re-RC-160), induced significantly increased tumor-growth inhibition compare with single 7.4 MBq 188Re-RC-160 treatment (P<.05).Conclusion: These studies showed that 188Re-RC-160 could be effectively used for targeting therapy the A549-derived tumors exogenously expressing hSSTR2, which will offers a potential therapeutic strategy for the treatment of somatostatin receptor-negative cancers.

Synthesis and in vivo brain distribution of carbon-11-labeled δ-opioid receptor agonists - Corrected Proof

2010-07-26

Abstract: Three new radiolabeled compounds, [11C]SNC80 ((+)-4-[(αR)-α-{(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl}-3-[11C]methoxybenzyl-N,N-diethylbenzamide), N,N-diethyl-4-[3-methoxyphenyl-1-[11C]methylpiperidin-4-ylidenemethyl)benzamide and N,N-diethyl-4-[(1-[11C]methylpiperidin-4-ylidene)phenylmethyl]benzamide, were prepared as potential in vivo radiotracers for the δ-opioid receptor. Each compound was synthesized by alkylation of the appropriate desmethyl compounds using [11C]methyl triflate. In vivo biodistribution studies in mice showed very low initial brain uptake of all three compounds and no regional specific binding for [11C]SNC80. A monkey positron emission tomography study of [11C]SNC80 confirmed low brain permeability and uniform regional distribution of this class of opioid agonists in a higher species. Opioid receptor ligands of this structural class are thus unlikely to succeed as in vivo radiotracers, likely due to efficient exclusion from the brain by the P-glycoprotein efflux transporter.

PEGylated N-methyl-S-methyl dithiocarbazate as a new reagent for the high-yield preparation of nitrido Tc-99m and Re-188 radiopharmaceuticals - Corrected Proof

2010-07-26

Abstract: A novel nitrido nitrogen atom donor for the preparation of 99mTc and 188Re radiopharmaceuticals containing a metal-nitrogen multiple bond is presented. HO2C-PEG600-DTCZ was obtained by conjugation of N-methyl-S-methyl dithiocarbazate [H2N–N(CH3)–C(S)SCH3, HDTCZ] with polyethylene glycol 600 (PEG600). Asymmetrical heterocomplexes of the type [M(N)(PNP)(B)]0/+ (M=99mTc, 188Re; PNP=diphosphine ligands, B=DBODC, DEDC, NSH, H2OS, CysNAc, HDTCZ) and symmetrical nitride compounds of the type [M(N)(L)2] (L=DEDC, DPDC) have been prepared in high yield by using the newly designed nitride nitrogen atom donor HO2C-PEG600-DTCZ. A two-step procedure was applied for preparing the above symmetrical and asymmetrical complexes. The first step involved the preliminary formation of a mixture of nitride Tc-99m or Re-188 precursors, which contained the [MN]2+ core, through reduction of generator-eluted 99mTc-pertechnetate or 188Re-perrhenate with thin (II) chloride in the presence of HO2C-PEG600-DTCZ. In the second step, the intermediate mixture was converted either in the final mixed asymmetrical complex by the simultaneous addition of diphosphine ligand and the suitable bidentate ligand B, or in the final symmetrical complex by the only addition of the bidentate ligand L. It was also demonstrated that the novel water-soluble nitride nitrogen atom donor HO2C-PEG600-DTCZ did not show coordinating properties toward the MN (99mTc, 188Re) core. Biodistribution studies in rats of the hitherto unreported [99mTc(N)(PNP3)DTCZ]+ and [99mTc(N)(PNP5)DTCZ]+ complexes showed that they selectively localize in the myocardium of rats with a favourable heart-to-lung and heart-to-liver uptake ratios. In particular, the heart-to-lung and heart-to-liver uptake ratios dramatically increased in the interval between 60 and 120 min postinjection. Hence, the combination of the favourable chemical and biological properties of HO2C-PEG600-DTCZ might confer to this novel compound an important role for the development of new 99mTc and 188Re-nitrido radiopharmaceuticals.

Comparison of l-type amino acid transporter 1 expression and l-[3-18F]-α-methyl tyrosine uptake in outcome of non-small cell lung cancer - Corrected Proof

2010-07-26

Abstract: Objective: l-Type amino acid transporter 1 (LAT1) has associated with tumor growth and poor outcome of patients with non-small cell lung cancer (NSCLC). l-[3-18F]-α-methyl tyrosine (18F-FAMT) is an amino acid tracer for positron emission tomography (PET) imaging, and 18F-FAMT uptake is mediated by LAT1. The purpose of this study is to compare the prognostic significance of 18F-FAMT uptake in the primary tumors with that of LAT1 expression in patients with NSCLC.Methods: Fifty-nine patients with NSCLC were enrolled in this study. All patients underwent 18F-FAMT PET prior to resection of the tumor, and immunohistochemical staining of the resected tumors were performed to compare the 18F-FAMT uptake and LAT1 expression. Uptake of 18F-FAMT was evaluated using semiquantitative standardized uptake value (SUVmax), and the cutoff value was determined to discriminate patients with high SUVmax from those with low SUVmax. Expression of LAT1 was evaluated by the score of staining intensity through 1 to 4. SUVmax and LAT1 expression were compared according to the clinicopathological variables.Results: The best discriminative cutoff value of 18F-FAMT SUVmax within the primary tumors was 1.6. The high SUVmax (>1.6) in 18F-FAMT PET was significantly associated with male, and positive LAT1 expression was significantly associated with male and nonadenocarcinoma. In the univariate analysis, high SUVmax (>1.6) in 18F-FAMT PET and positive LAT1 expression were significant predictor of the poor outcome. Multivariate analysis confirmed that positive LAT1 expression was an independent and significant factor for predicting poor prognosis in NSCLC (P=.035).Conclusion: LAT1 expression is a stronger prognostic factor than 18F-FAMT uptake in surgically resected NSCLC.

Multivalent cyclic RGD ligands: influence of linker lengths on receptor binding - Corrected Proof

2010-07-26

Abstract: Peptides involving the RGD motive (arginine–glycine–aspartic acid) recognize members of the integrin receptor family. Since the receptors are located mainly on the surface of endothelial cells, structural modifications including multimers of c(RGDfE) were recently found to improve the binding avidity for αvβ3 integrin significantly. The multivalent RGD peptides exhibited rather loose linkages partly including oligo(ethylene glycol) spacers (EGn) with different chain lengths. Therefore, the dependence of multivalent RGD systems with and without EGn linkers were investigated on their binding properties to cultured αvβ3 integrin-expressing U87MG cells.Methods: We synthesized a series of di-, tri- and tetravalent rigid scaffolds (terephthalic acid, trimesic acid and adamantane-1,3,5,7-tetracarboxylic acid) conjugated to c(RGDyK) ligands, which were linked contiguously or separated by the oligo(ethylene glycol) spacers. The inhibition constants of these c(RGDyK) derivatives were determined by competition assays with 125I-labeled echistatin.Results: While c(RGDyK) function is a relative weak competitor against [125I]echistatin (Ki, 329±18 nM) for αvβ3 integrin-expressing U87MG cells, RGD dimers improved the competition potency considerably (Ki, 64±23 nM). This effect was even more pronounced with the RGD trimers (Ki, 40±7 nM) and tetramers (Ki, 26±9 nM). The introduction of EGn spacers and the increase of linker lengths proved to be detrimental since more competitors were needed to compete with [125I]echistatin. The EG6 group, for example, reduced the inhibition constants by 29% (dimer), 57% (trimer) and 97% (tetramer).Conclusion: The binding experiments performed with the three forms of multivalent RGD ligands indicate the weakening of competitive potency against [125I]echistatin with the introduction of EGn spacers. This effect may be related to the decrease of the effective RGD molarity, which becomes most prominent within the tetravalent series.

Radioiodinated 4-iodo-l-meta-tyrosine, a system L selective artificial amino acid: molecular design and transport characterization in Chinese hamster ovary cells (CHO-K1 cells) - Corrected Proof

2010-07-26

Abstract: Introduction: High expression of the system L amino acid transporter has been observed in clinically important tissues including tumors and the blood-brain barrier. We examined amino acid transport system L selectivity of 14C(U)-l-tyrosine (14C-Tyr), 125I-4-iodo-l-meta-tyrosine (4-125I-mTyr), 125I-6-iodo-l-meta-tyrosine (6-125I-mTyr), 125I-3-iodo-α-methyl-l-tyrosine (125I-IMT) and 125I-3-iodo-l-tyrosine (3-125I-Tyr) using Chinese hamster ovary cells (CHO-K1).Methods: Cells in the exponential growth phase were incubated with 18.5 kBq of labeled amino acid in 2 mL of phosphate-buffered saline-based uptake solution and an uptake solution with/without Na+ at 37°C or 4°C. We examined the effects of the following compounds (1.0 mM) on transport: 2-(methylamino)isobutyric acid (a specific inhibitor of system A, in Na+-containing uptake solution); 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (a specific inhibitor of system L, in Na+-free uptake solution); sodium azide and 2,4-dinitrophenol (NaN3 and DNP, inhibitors of the generation of adenosine triphosphate); p-aminohippurate and tetraethylammonium (PAH and TEA, inhibitors of organic anion and cation transporters); and l- and d-isomers of natural amino acids.Results: 14C-Tyr exhibited affinity for systems L, A and ASC. 4-125I-mTyr and 3-125I-Tyr exhibited high specificity for system L, whereas 6-125I-mTyr and 125I-IMT exhibited affinity for both systems L and ASC. Uptake of 4-125I-mTyr was markedly reduced by incubation at 4 °C, and was not significantly inhibited by NaN3, DNP, PAH or TEA. The inhibition profiles of the l- and d-isomers of natural amino acids indicated that system L mediates the transport of 4-125I-mTyr.Conclusions: 4-125I-mTyr exhibited the greatest system L specificity (93.46±0.13%) of all of the tested amino acids.

Evaluation of a bolus/infusion protocol for 11C-ABP688, a PET tracer for mGluR5 - Corrected Proof

2010-06-30

Abstract: 11C-ABP-688 is a selective tracer for the mGluR5 receptor. Its kinetics is fast and thus favourable for an equilibrium approach to determine receptor-related parameters. The purpose of this study was to test the hypothesis that the pattern of the 11C-ABP688 uptake using a bolus-plus-infusion (B/I) protocol at early time points corresponds to the perfusion and at a later time point to the total distribution volume.Methods: A bolus and a B/I study (1 h each) was performed in five healthy male volunteers. With the B/I protocol, early and late scans were normalized to gray matter, cerebellum and white matter. The same normalization was done on the maps of the total distribution volume (Vt) and K1 which were calculated in the study with bolus only injection and the Logan method (Vt) and a two-tissue compartment model (K1).Results: There was an excellent correlation close to the identity line between the pattern of the late uptake in the B/I study and Vt of the bolus-only study for all three normalizations. The pattern of the early uptake in the B/I study correlated well with the K1 maps, but only when normalized to gray matter and cerebellum, not to white matter.Conclusion: It is demonstrated that with a B/I protocol the 11C-ABP688 distribution in late scans reflects the pattern of the total distribution volume and is therefore a measure for the density pattern of mGluR5. The early scans following injection are related to blood flow, although not in a fully quantitative manner. The advantage of the B/I protocol is that no arterial blood sampling is required, which is advantageous in clinical studies.

Optimization of automated radiosynthesis of [18F]AV-45: a new PET imaging agent for Alzheimer's disease - Corrected Proof

2010-06-30

Abstract: Introduction: Accumulation of β-amyloid (Aβ) aggregates in the brain is linked to the pathogenesis of Alzheimer's disease (AD). Imaging probes targeting these Aβ aggregates in the brain may provide a useful tool to facilitate the diagnosis of AD. Recently, [18F]AV-45 ([18F]5) demonstrated high binding to the Aβ aggregates in AD patients. To improve the availability of this agent for widespread clinical application, a rapid, fully automated, high-yield, cGMP-compliant radiosynthesis was necessary for production of this probe. We report herein an optimal [18F]fluorination, de-protection condition and fully automated radiosynthesis of [18F]AV-45 ([18F]5) on a radiosynthesis module (BNU F-A2).Methods: The preparation of [18F]AV-45 ([18F]5) was evaluated under different conditions, specifically by employing different precursors (-OTs and -Br as the leaving group), reagents (K222/K2CO3 vs. tributylammonium bicarbonate) and deprotection in different acids. With optimized conditions from these experiments, the automated synthesis of [18F]AV-45 ([18F]5) was accomplished by using a computer-programmed, standard operating procedure, and was puriﬁed on an on-line solid-phase cartridge (Oasis HLB).Results: The optimized reaction conditions were successfully implemented to an automated nucleophilic fluorination module. The radiochemical purity of [18F]AV-45 ([18F]5) was >95%, and the automated synthesis yield was 33.6±5.2% (no decay corrected, n=4), 50.1±7.9% (decay corrected) in 50 min at a quantity level of 10–100 mCi (370–3700 MBq). Autoradiography studies of [18F]AV-45 ([18F]5) using postmortem AD brain and Tg mouse brain sections in the presence of different concentration of “cold” AV-136 showed a relatively low inhibition of in vitro binding of [18F]AV-45 ([18F]5) to the Aβ plaques (IC50=1–4 μM, a concentration several order of magnitude higher than the expected pseudo carrier concentration in the brain).Conclusions: Solid-phase extraction purification and improved labeling conditions were successfully implemented into an automated synthesis module, which is more convenient, highly efficient and simpler in operation than using a semipreparative high-performance liquid chromatography method. This new, automated procedure for preparation of [18F]AV-45 ([18F]5) is suitable for routine clinical application.

Evaluation of anti-podoplanin rat monoclonal antibody NZ-1 for targeting malignant gliomas - Corrected Proof

2010-06-07

Abstract: Introduction: Podoplanin/aggrus is a mucin-like sialoglycoprotein that is highly expressed in malignant gliomas. Podoplanin has been reported to be a novel marker to enrich tumor-initiating cells, which are thought to resist conventional therapies and to be responsible for cancer relapse. The purpose of this study was to determine whether an anti-podoplanin antibody is suitable to target radionuclides to malignant gliomas.Methods: The binding affinity of an anti-podoplanin antibody, NZ-1 (rat IgG2a), was determined by surface plasmon resonance and Scatchard analysis. NZ-1 was radioiodinated with 125I using Iodogen [125I-NZ-1(Iodogen)] or N-succinimidyl 4-guanidinomethyl 3-[131I]iodobenzoate ([131I]SGMIB-NZ-1), and paired-label internalization assays of NZ-1 were performed. The tissue distribution of 125I-NZ-1(Iodogen) and that of [131I]SGMIB-NZ-1 were then compared in athymic mice bearing glioblastoma xenografts.Results: The dissociation constant (KD) of NZ-1 was determined to be 1.2×10−10 M by surface plasmon resonance and 9.8×10−10 M for D397MG glioblastoma cells by Scatchard analysis. Paired-label internalization assays in LN319 glioblastoma cells indicated that [131I]SGMIB-NZ-1 resulted in higher intracellular retention of radioactivity (26.3±0.8% of initially bound radioactivity at 8 h) compared to that from the 125I-NZ-1(Iodogen) (10.0±0.1% of initially bound radioactivity at 8 h). Likewise, tumor uptake of [131I]SGMIB-NZ-1 (39.9±8.8 %ID/g at 24 h) in athymic mice bearing D2159MG xenografts in vivo was significantly higher than that of 125I-NZ-1(Iodogen) (29.7±6.1 %ID/g at 24 h).Conclusions: The overall results suggest that an anti-podoplanin antibody NZ-1 warrants further evaluation for antibody-based therapy against glioblastoma.

Fluorine-18 radiopharmaceuticals beyond [18F]FDG for use in oncology and neurosciences - Corrected Proof

2010-06-07

Abstract: Positron emission tomography (PET) is a rapidly expanding clinical modality worldwide thanks to the availability of compact medical cyclotrons and automated chemistry for the production of radiopharmaceuticals. There is an armamentarium of fluorine-18 (18F) tracers that can be used for PET studies in the fields of oncology and neurosciences. However, most of the 18F-tracers other than 2-deoxy-2-[18F]fluoro-D-glucose (FDG) are in less than optimum human use and there is considerable scope to bring potentially useful 18F-tracers to clinical investigation stage.The International Atomic Energy Agency (IAEA) convened a consultants' group meeting to review the current status of 18F-based radiotracers and to suggest means for accelerating their use for diagnostic applications. The consultants reviewed the developments including the synthetic approaches for the preparation of 18F-tracers for oncology and neurosciences. A selection of three groups of 18F-tracers that are useful either in oncology or in neurosciences was done based on well-defined criteria such as application, lack of toxicity, availability of precursors and ease of synthesis. Based on the recommendations of the consultants' group meeting, IAEA started a coordinated research project on “Development of 18F radiopharmaceuticals (beyond [18F]FDG) for use in oncology and neurosciences” in which 14 countries are participating in a 3-year collaborative program. The outcomes of the coordinated research project are expected to catalyze the wider application of several more 18F-radiopharmaceuticals beyond FDG for diagnostic applications in oncology and neurosciences.

The first design and synthesis of [11C]MKC-1 ([11C]Ro 31-7453), a new potential PET cancer imaging agent - Corrected Proof

2010-06-07

Abstract: Bisindolylmaleimide MKC-1 (formerly known as Ro 31-7453) is a novel, orally active, small-molecule, cell cycle inhibitor with broad-spectrum antitumor effects. [11C]MKC-1 ([11C]Ro 31-7453) was first designed and synthesized as a new potential positron emission tomography cancer imaging agent through two different strategies. The first strategy was to prepare a carbon-11-labeled bisindolyl maleic anhydride intermediate followed by the conversion to maleimide. The second strategy involved the condensation of either carbon-11-labeled indole-3-acetamides with indole-3-glyoxalates, or indole-3-acetamides with carbon-11-labeled indole-3-glyoxalates. The radiochemical yields were 15–30%, decay corrected to end of bombardment (EOB), based on [11C]CO2. The specific activity was 222–296 GBq/μmol at EOB and 111–148 GBq/μmol at the end of synthesis, respectively.

Systemic catechol-O-methyl transferase inhibition enables the D1 agonist radiotracer R-[11C]SKF 82957 - Corrected Proof

Mikael Palner, Patrick McCormick, Jun Parkes, Gitte M. Knudsen, Alan A. Wilson — 2010-06-07

Abstract: Introduction: R-[11C]-SKF 82957 is a high-affinity and potent dopamine D1 receptor agonist radioligand, which gives rise to a brain-penetrant lipophilic metabolite. In this study, we demonstrate that systemic administration of catechol-O-methyl transferase (COMT) inhibitors blocks this metabolic pathway, facilitating the use of R-[11C]-SKF 82957 to image the high-affinity state of the dopamine D1 receptor with PET.Methods: R-[11C]SKF 82957 was administered to untreated and COMT inhibitor-treated conscious rats, and the radioactive metabolites present in the brain and plasma were quantified by HPLC. Under optimal conditions, cerebral uptake and dopamine D1 binding of R-[11C]SKF 82957 were measured ex vivo. In addition, pharmacological challenges with the receptor antagonist SCH 23390, amphetamine, the dopamine reuptake inhibitor RTI-32 and the dopamine hydroxylase inhibitor α-methyl-p-tyrosine were performed to study the specificity and sensitivity of R-[11C]-SKF 82957 dopamine D1 binding in COMT-inhibited animals.Results: Treatment with the COMT inhibitor tolcapone was associated with a dose-dependent (EC90 5.3±4.3 mg/kg) reduction in the lipophilic metabolite. Tolcapone treatment (20 mg/kg) also resulted in a significant increase in the striatum/cerebellum ratio of R-[11C]SKF 82957, from 15 (controls) to 24. Treatment with the dopamine D1 antagonist SCH 23390 reduced the striatal binding to the levels of the cerebellum, demonstrating a high specificity and selectivity of R-[11C]SKF 82957 binding.Conclusions: Pre-treatment with the COMT inhibitor tolcapone inhibits formation of an interfering metabolite of R-[11C]SKF 82957. Under such conditions, R-[11C]SKF 82957 demonstrates high potential as the first agonist radiotracer for imaging the dopamine D1 receptor by PET.

In vivo SPECT/CT imaging of human orthotopic ovarian carcinoma xenografts with 111In-labeled monoclonal antibodies - Corrected Proof

2010-05-31

Abstract: Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 3 (VEGFR-3) are expressed in the tumor area during the progression of ovarian carcinoma. Monoclonal antibodies developed against these receptors are potential diagnostic molecules for in vivo imaging of ovarian carcinoma.Methods: Biodistribution of the monoclonal antibodies cetuximab against EGFR and mF4-31C1 against VEGFR-3 was studied in nude mice with orthotopic SKOV-3m human ovarian carcinoma xenografts. The biodistribution of 111Indium-labeled antibodies was followed up to 48 h postinjection using combined SPECT and CT imaging modality. Organ samples were collected postmortem and specific organ activity was measured. Accumulation of the intravenously injected antibodies in the tumor tissue and lymph nodes was verified using immunohistology.Results: Imaging studies with SPECT/CT showed clear accumulation of both antibodies into tumor area. The tumor uptake was 8.78±0.74 %ID/g for cetuximab and 5.77±0.62 %ID/g for mF4-31C1 after 48 h postinjection. Cetuximab had lower liver tropism and faster tumor homing rate. In addition, after 48 h two of five tumor-bearing mice showed a clear accumulation of the In-labeled mF4-31C1 at the left axillary area. Both intravenously administered antibodies could also be detected from the tumor sections by immunohistological staining but only mF4-31C1 forms in the lymph nodes.Conclusion: These results demonstrate the accumulation of EGFR- and VEGFR-3-specific antibodies in orthotopic ovarian carcinoma tumors. Systemically administered they had slow pharmacokinetics which is typical for antibodies. Accumulation of mF4-31C1 antibody in the lymph nodes suggests the remote activation of VEGFR-3 by the primary tumor.

The effect of radiocolloid preference on major parameters in sentinel lymph node biopsy practice in breast cancer - Corrected Proof

Ulkem Yararbas, A. Murat Argon, Levent Yeniay, Baha Zengel, Murat Kapkaç — 2010-05-31

Abstract: Introduction: The possible effects of radiocolloid preference on sentinel lymph node biopsy (SLNB) were investigated.Methods: A total of 200 patients with T1-2N0M0 breast cancer were evaluated. The first 100 patients underwent SLNB using 99mTc tin colloid (TC) and the next 100 using 99mTc nanocolloid (NC). Radiocolloid was injected intradermally at four quadrants of the periareolar region the day before surgery. All patients underwent lymphoscintigraphy 1 h after injection. All nodes having fourfold activity of the background were harvested using gamma probe.Results: Sentinel lymph node (SLN) identification rate by gamma probe was 98% in each group. The number of SLNs identified by lymphoscintigraphy, gamma probe and pathological evaluation was 1.39±0.7, 1.70±1.0 and 2.23±1.70 in the TC and 2.03±0.94, 2.60±1.36 and 3.05±1.90 in the NC group, respectively (P<.05). Metastatic SLN was found in 24 (24.4%) of 98 patients in the TC group and 41 (41.8%) of 98 patients in the NC group (P=.04). None of the patients showed dispersion to internal mammarian lymph nodes. Lymphatic vessel visualization was observed in eight (8.1%) of 98 TC patients and in 47 (47.9%) of 98 NC patients (P=.000). SLNs were the only metastatic node(s) in 54.1% of TC and 73.1% of NC patients.Conclusion: The periareolar intradermal injection technique gives a high detection rate in the localization of SLNs independently from the choice of the tracer. Mean SLN numbers and lymphatic vessel visualization frequency were significantly higher using a smaller albumin Tc-99m nanocolloid as compared to a stannous fluoride Tc-99m tin colloid. The results of our study support the idea that the influence of increased number of SLNs on positive SLN frequency is critical.

Investigation of 99mTc-labelling of recombinant human interleukin-2 via hydrazinonicotinamide - Corrected Proof

2010-05-31

Abstract: Introduction: Interleukin-2 (IL-2) when radiolabelled with 99mTc has been proved useful in imaging the side of lymphocytic infiltration in patients with autoimmune disorders and plays a significant role as a T-cell imaging agent. However, the labelling procedures used so far appeared to be rather complex and laborious. The aim of present study was to develop an efficient procedure of 99mTc-labelling of recombinant human interleukin-2 (rhIL-2) via hydrazinonicotinamide (HYNIC) to develop a dry kit formulation.Methods: Various molar ratios of rhIL-2/HYNIC (from 1:2 to 1:12) were used at the conjugation step. The conjugates were purified on a PD-10 column to remove the excess of unbound HYNIC, as well as of any aggregates. The final peptide concentration was quantified by the BCA method, and the number of HYNIC molecules incorporated into a rhIL-2 molecule was determined based on the reaction with 2-sulfobenzaldehyde. The 99mTc-labelling was optimized using various amounts of HYNIC–rhIL-2, 99mTc, SnCl2, tricine and nicotinic acid (NA). Quality control included GF-HPLC, ITLC, SDS-PAGE and biological assay. Biodistribution studies were performed in Swiss mice and Wistar rats.Results: Generally, the highest radiolabelling yields were achieved when the HYNIC–rhIL-2 conjugates of ca. 2–4 HYNIC molecule substitution ratios were used. The optimal pH of the reaction medium was found to be in the range of 6.5 to 7.0. GF-HPLC analysis indicated that monomer and aggregates of 99mTc-HYNIC–rhIL-2 are formed during radiolabelling. At optimized conditions of wet radiolabelling, the 99mTc-HYNIC–rhIL-2 monomer was obtained with radiochemical purity >99%, specific activity of ca. 4 GBq/mg rhIL-2 and overall yield of ca. 65%. The two-vial freeze-dried kit was prepared: the first vial contained 30 μg HYNIC–rhIL-2, co-ligands, buffer and antioxidant; the second vial contained tricine and SnCl2. The monomer of 99mTc-HYNIC–rhIL-2 was obtained by gel chromatography on a PD-10 column. No differences between labelled and unlabelled IL2 in terms of biological activity were observed.Conclusions: Our study shows that rhIL-2 can be efficiently radiolabelled with 99mTc via HYNIC, with tricine and NA as co-ligands using a two-vial freeze-dried kit. This enables the preparation of sterile and ready-to-use 99mTc-HYNIC(tricine,NA)-rhIL-2 within 1 h.

Optimization, biological evaluation and microPET imaging of copper-64-labeled bombesin agonists, [64Cu-NO2A-(X)-BBN(7–14)NH2], in a prostate tumor xenografted mouse model - Corrected Proof

2010-05-31

Abstract: Gastrin-releasing peptide receptors (GRPr) are a member of the bombesin (BBN) receptor family. GRPr are expressed in high numbers on specific human cancers, including human prostate cancer. Therefore, copper-64 (64Cu) radiolabeled BBN(7–14)NH2 conjugates could have potential for diagnosis of human prostate cancer via positron-emission tomography (PET). The aim of this study was to produce [64Cu-NO2A-(X)-BBN(7–14)NH2] conjugates for prostate cancer imaging, where X=pharmacokinetic modifier (beta-alanine, 5-aminovaleric acid, 6-aminohexanoic acid, 8-aminooctanoic acid, 9-aminonanoic acid or para-aminobenzoic acid) and NO2A=1,4,7-triazacyclononane-1,4-diacetic acid [a derivative of NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid)].Methods: [(X)-BBN(7–14)NH2] Conjugates were synthesized by solid-phase peptide synthesis (SPPS), after which NOTA was added via manual conjugation. The new peptide conjugates were radiolabeled with 64Cu radionuclide. The receptor-binding affinity was determined in human prostate PC-3 cells, and tumor-targeting efficacy was determined in PC-3 tumor-bearing severely combined immunodeficient (SCID) mice. Whole-body maximum intensity microPET/CT images of PC-3 tumor-bearing SCID mice were obtained 18 h postinjection (pi).Results: Competitive binding assays in PC-3 cells indicated high receptor-binding affinity for the [NO2A-(X)-BBN(7–14)NH2] and [natCu-NO2A-(X)-BBN(7–14)NH2] conjugates. In vivo biodistribution studies of the [64Cu-NO2A-(X)-BBN(7–14)NH2] conjugates at 1, 4 and 24 h pi showed very high uptake of the tracer in GRPr-positive tissue with little accumulation and retention in nontarget tissues.High-quality, high-contrast microPET images were obtained, with xenografted tumors being clearly visible at 18 h pi.Conclusions: NO2A chelator sufficiently stabilizes copper(II) radiometal under in vivo conditions, producing conjugates with very high uptake and retention in targeted GRPr. Preclinical evaluation of these new peptide conjugates in tumor-bearing mice provides some impetus for clinical evaluation in human patients.

Anti-EGFRvIII monoclonal antibody armed with 177Lu: in vivo comparison of macrocyclic and acyclic ligands - Corrected Proof

2010-05-31

Abstract: Introduction: Monoclonal antibody (mAb) L8A4 binds specifically to the epidermal growth factor receptor variant III (EGFRvIII) that is present on gliomas but not on normal tissues, and is internalized rapidly after receptor binding. Because of the short range of its β-emissions, labeling this mAb with 177Lu would be an attractive approach for the treatment of residual tumor margins remaining after surgical debulking of brain tumors.Materials and Methods: L8A4 mAb was labeled with 177Lu using the acyclic ligands [(R)-2-amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid (CHX-A″-DTPA) and 2-(4-isothiocyanatobenzyl)-6-methyldiethylene-triaminepentaacetic acid (1B4M-DTPA), and the macrocyclic ligands S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-tetraacetic acid (C-DOTA) and α-(5-isothiocyanato-2-methoxyphenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (MeO-DOTA). Paired-label tissue distribution experiments were performed in athymic mice bearing subcutaneous EGFRvIII-expressing U87.ΔEGFR glioma xenografts over a period of 1 to 8 days to directly compare 177Lu-labeled L8A4 to L8A4 labeled with 125I using N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB).Results: Except with C-DOTA, tumor uptake for the 177Lu-labeled mAb was significantly higher than the co-administered radioiodinated preparation; however, this was also the case for spleen, liver, bone and kidneys. Tumor/normal tissue ratios for 177Lu-1B4M-DTPA-L8A4 and, to an even greater extent, 177Lu-MeO-DOTA-L8A4 were higher than those for [125I]SGMIB-L8A4 in most other tissues.Conclusions: Tumor and normal tissue distribution patterns for this anti-EGFRvIII mAb were dependent on the nature of the bifunctional chelate used for 177Lu labeling. Optimal results were obtained with 1B4M-DTPA and MeO-DOTA, suggesting no clear advantage for acyclic vs. macrocyclic ligands for this application.

Increased Lipiodol uptake in hepatocellular carcinoma possibly due to increased membrane fluidity by dexamethasone and tamoxifen - Corrected Proof

2010-05-31

Abstract: Introduction: Lipiodol is used as a vector for chemoembolization or internal radiotherapy in unresectable hepatocellular carcinomas (HCCs). The aim of this study is to improve the tumoral uptake of Lipiodol by modulating membrane fluidizing agents to optimize the effectiveness of Lipiodol vectorized therapy.Methods: The effect of dexamethasone and tamoxifen on membrane fluidity was studied in vitro by electron paramagnetic resonance applied to rat hepatocarcinoma cell line N1S1.The tumoral uptake of Lipiodol was studied in vivo on rats with HCC, which had been previously treated by dexamethasone and/or tamoxifen, after intra-arterial administration of 99mTc-SSS-Lipiodol.Results: The two molecules studied here exhibit a fluidizing effect in vitro which appears dependent on time and dose, with a maximum fluidity obtained after 1 hr at concentrations of 20 μM for dexamethasone and 200 nM for tamoxifen. In vivo, while the use of dexamethasone or tamoxifen alone tends to lead to increased tumoral uptake of Lipiodol, this effect does not reach levels of significance. On the other hand, there is a significant increase in the tumoral uptake of 99mTc-SSS-Lipiodol in rats pretreated by both dexamethasone and tamoxifen, with a tumoral uptake (expressed in % of injected activity per g of tumor) of 13.57±3.65% after treatment, as against 9.45±4.44% without treatment (P<.05).Conclusions: Dexamethasone and tamoxifen fluidify the N1S1 cells membrane, leading to an increase in the tumoral uptake of Lipiodol. These drugs could be combined with chemo-Lipiodol-embolization or radiolabeled Lipiodol, with a view to improving the effectiveness of HCCs therapy.

An electro-amalgamation approach to isolate no-carrier-added 177Lu from neutron irradiated Yb for biomedical applications - Corrected Proof

Rubel Chakravarty, Tapas Das, Ashutosh Dash, Meera Venkatesh — 2010-05-31

Abstract: Introduction: A novel two-step separation process for the production of no-carrier-added (NCA) 177Lu from neutron irradiated Yb target through an electrochemical pathway employing mercury-pool cathode has been developed.Methods: A two-cycle electrolysis procedure was adopted for separation of 177Lu from 177Lu/Yb mixture in lithium citrate medium. The influence of different experimental parameters on the separation process was investigated and optimized for the quantitative deposition of Yb in presence of 177Lu. The first electrolysis was performed for 50 min in the 177Lu/Yb feed solution at pH 6 applying a potential of 8 V using platinum electrode as anode and mercury as the cathode. The second electrolysis was performed under the same conditions using fresh electrodes. The radionuclidic and chemical purity of 177Lu was determined by using gamma ray spectrometry and atomic absorption spectrometry. The suitability of 177Lu for biomedical applications was ascertained by labeling 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid D-Phe1-Tyr3-octreotate(DOTA-TATE) with 177Lu.Results: This process could provide NCA 177Lu with >99.99% radionuclidic purity and an overall separation yield of ∼99% was achieved within 3–4 h. The Hg content in the product was determined to be <1 ppm. Radiolabeling yield of >98% was obtained with DOTA-TATE under the optimized reaction conditions.Conclusions: An efficient strategy for the separation of NCA 177Lu, suitable for biomedical applications, has been developed.

Measurement of dopamine D2 receptors in living human brain using [11C]raclopride with ultra-high specific radioactivity - Corrected Proof

2010-05-31

Abstract: Introduction: High specific radioactivity is preferable in the measurement of neuroreceptor bindings with positron emission tomography (PET) because receptor occupancy by mixed cold ligand hampers the accurate estimation of receptor binding. Recently, we succeeded in synthesizing [11C]raclopride, a dopamine D2 receptor ligand, with ultra-high specific radioactivity, i.e., several thousand GBq/μmol. In the present study, we compared the [11C]raclopride bindings to dopamine D2 receptors between radioligands with ultra-high specific radioactivity and ordinary high specific radioactivity in healthy human subjects.Methods: Two PET studies using [11C]raclopride with ultra-high specific radioactivity (4302–7222 GBq/μmol) or ordinary high specific radioactivity (133-280 GBq/μmol) were performed on different days in 14 healthy men. Binding potential (BP) was calculated by the simplified reference tissue method, peak equilibrium method, and area-under-the-curve method for each region-of-interest using time-activity data in the cerebellum as a reference brain region.Results: BP values for radioligands with ultra-high specific radioactivity and ordinary high specific radioactivity calculated by the simplified reference tissue method were 4.06±0.29 and 4.10±0.25 in the putamen, 0.44±0.07 and 0.47±0.07 in the thalamus and 0.37±0.06 and 0.38±0.06 in the temporal cortex, respectively (mean±S.D.). No significant difference in BP was observed between ultra-high specific radioactivity and ordinary high specific radioactivity in any of the brain regions.Conclusion: BP values of [11C]raclopride with ultra-high specific radioactivity did not differ from those with ordinary high specific radioactivity in the measured brain regions, including striatal and extrastriatal regions.

Synthesis and evaluation of 99mTc-N-sulfanilamide ferrocene carboxamide as bacterial infections detector - Corrected Proof

Imen Essouissi, Wafa Ghali, Nadia Malek Saied, Mouldi Saidi — 2010-05-31

Abstract: A technetium-99m-labeled derivative from sulfanilamide, further referred to as 99mTc-N-SFC, targeting infections in experimental animals, has been synthesized. The biological features of this radioactive agent have also been studied. The N-sulfanilamide ferrocene carboxamide (N-SFC) was chemically synthesized and then labeled with technetium-99m. It has been confirmed through this work that it is stable and obtained with radiolabelling yield (>87%). Radiochemical analyses of 99mTc-N-SFC revealed that the molecule was labeled rapidly (within 2 min) and effectively with little free pertechnetate in the preparations containing purified compound. Furthermore, in vitro investigations were conducted and the label's stability in serum was observed up to 24 h of testing. Uptake of the tracer with live and heat/killed bacteria was compared in physiological conditions and was about 69% and 61.9% for the Escherichia coli and Staphylococcus aureus strains, respectively. We concluded that synthesis and labeling of Sulfanilamide derivative with 99m-Tc by this method is rapid, efficient and safe. Biodistribution studies demonstrated that our radiolabeled compound is accumulated rapidly and significantly (P<.05) at infection sites. The comparison of the 99mTc-N-SFC accumulation at sites of S. aureus-infected animals, which is expressed as target-to-non-target ratio, (2.88±0.10) with other radiotracers was discussed.

In vivo imaging and quantitative analysis of TSPO in rat peripheral tissues using small-animal PET with [18F]FEDAC - Corrected Proof

2010-05-31

Abstract: Introduction: The translocator protein (18 kDa) (TSPO) is widely expressed in peripheral tissues, including the heart, lung, and kidney. Our laboratory developed N-benzyl-N-methyl-2-[7,8-dihydro-7-(2-[18F]fluoroethyl)-8-oxo-2-phenyl-9H-purin-9-yl]acetamide ([18F]FEDAC) as a TSPO positron emission tomography (PET) ligand. Here, using small-animal PET with [18F]FEDAC, we performed TSPO imaging and quantitative analysis of TSPO binding in rat peripheral tissues.Methods: The in vivo distribution and kinetics of [18F]FEDAC were measured in rat peripheral tissues (heart, lung and kidney). Using the in vivo pseudo-equilibrium method, TSPO binding parameters [TSPO density (Bmax), dissociation constant (KD)] and receptor occupancy were estimated in these peripheral tissues.Results: [18F]FEDAC was highly distributed in the lung, heart and kidney, and these TSPO-enriched tissues could be clearly visualized. The kinetics of this radioligand in these tissues was rapid, which is suitable for the determination of in vivo TSPO binding parameters and receptor occupancy. The Bmax value of TSPO in the heart, lung, and kidney was 393, 141, and 158 pmol/ml, respectively. The KD value of the radioligand in the heart, lung, and kidney was 119, 36 and 123 nM, respectively. By pretreatment with 5 mg/kg Ro 5-4864 (a TSPO ligand), about 90% of binding sites for TSPO in the heart and lung were occupied. In the kidney, the binding sites were completely occupied by 5 mg/kg Ro 5-4864.Conclusions: [18F]FEDAC is a suitable PET ligand for TSPO imaging and quantitative analysis of TSPO binding in rat peripheral tissues. The utilization of [18F]FEDAC-PET and the pseudo-equilibrium method can contribute to the study of the TSPO function and evaluate the in vivo binding parameters and receptor occupancy of TSPO therapeutic compounds.