Peter Laverman

A Novel Method of 18F Radiolabeling for PET

Small biomolecules are typically radiolabeled with 18F by binding it to a carbon atom, a process that usually is designed uniquely for each new molecule and requires several steps and hours to produce. We report a facile method wherein 18F is first attached to aluminum as Al18F, which is then bound to a chelate attached to a peptide, forming a stable Al18F-chelate-peptide complex in an efficient 1-pot process. Methods: For proof of principle, this method was applied to a peptide suitable for use in a bispecific antibody pretargeting method. A solution of AlCl3·6H2O in a pH 4.0 sodium-acetate buffer was mixed with an aqueous solution of 18F to form the Al18F complex. This was added to a solution of IMP 449 (NOTA-p-Bn-CS-d-Ala-d-Lys(HSG)-d-Tyr-d-Lys(HSG)-NH2) (NOTA-p-Bn-CS is made from S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid; HSG is histamine-succinyl-glycine) and heated to 100°C for 15 min. In vitro and in vivo stability and targeting ability of the Al18F-IMP 449 were examined in nude mice bearing LS174T human colonic tumors pretargeted with an anti-CEACAM5 bispecific antibody (TF2). Results: The radiolabeled peptide was produced in 5%−20% yield with an estimated specific activity of 18,500–48,100 GBq (500–1,300 Ci)/mmol. The Al18F-IMP 449 was stable for 4 h in serum in vitro, and in animals, activity isolated in the urine 30 min after injection was bound to the peptide. Nonchelated Al18F had higher tissue uptake, particularly in the bones, than the chelated Al18F-IMP 449, which cleared rapidly from the body by urinary excretion. Tumor uptake was 30-fold higher with TF2-pretargeted Al18F-IMP 449 than with the peptide alone. Dynamic PET showed tumor localization within 30 min and rapid and thorough clearance from the body. Conclusion: The ability to bind highly stable Al18F to metal-binding ligands is a promising new labeling method that should be applicable to a diverse array of molecules for PET.

Fluorinated amino acids for tumour imaging with positron emission tomography

Abstract. The currently preferred radiopharmaceutical for positron emission tomography (PET) in oncology is 2-[18F]fluoro-deoxyglucose (FDG). Increased accumulation of this deoxyglucose analogue in tumour cells is based on elevated glucose metabolism by tumour cells and subsequent trapping in the cells. In the search for new PET tracers, amino acids have been widely studied. A new amino acid tracer should preferably have similar high uptake in tumour cells but low non-specific uptake in normal tissues and any pathology other than tumour. In recent years, several amino acids have been labelled with either gamma radiation-emitting radionuclides or positron-emitting radionuclides, the most commonly used being carbon-11. However, the longer half-life of fluorine-18 matches better with the relatively slow process of protein synthesis and also facilitates shipping of the radiolabelled amino acids to hospitals without an on-site cyclotron or dedicated radiochemistry laboratory. The number of fluorinated amino acids under investigation is increasing, and one of the major points of discussion is the underlying mechanism of the tumour visualisation. While it has been shown that some amino acids can be used to measure the protein synthesis rate, others are used with the sole aim of measuring the rate of uptake into the cell. The differences between measuring amino acid transport (rate) and protein synthesis rate with 18F-labelled amino acids are discussed.

Spatial Resolution and Sensitivity of the Inveon Small-Animal PET Scanner

The Inveon small-animal PET scanner is characterized by a large, 127-mm axial length and a 161-mm crystal ring diameter. The associated high sensitivity is obtained by using all lines of response (LORs) up to the maximum ring difference (MRD) of 79, for which the most oblique LORs form acceptance angles of 38.3° with transaxial planes. The result is 2 phenomena that are normally not encountered in PET scanners: a parallax or depth-of-interaction effect in the axial direction and the breakdown of Fourier rebinning (FORE). Both effects cause a deterioration of axial spatial resolution. Limiting the MRD to smaller values reduces this axial blurring at the cost of sensitivity. Alternatively, 3-dimensional (3D) reconstruction techniques can be used in which the rebinning step is absent. The aim of this study was to experimentally determine the spatial resolution and sensitivity of the Inveon for its whole field of view (FOV). Methods: Spatial resolution and sensitivity were measured using filtered backprojection (FBP) with FORE, FBP with LOR angle-weighted adapted FORE (AFORE), and 3D ordered-subset expectation maximization followed by maximum a posteriori reconstruction (OSEM3D/MAP). Results: Tangential and radial full width at half maximum (FWHM) showed almost no dependence on the MRD using FORE and FBP. Tangential FWHMs were 1.5 mm in the center of the FOV (CFOV) and 1.8 mm at the edge of the FOV (EFOV). Radial FWHMs were 1.5 and 3.0 mm in the CFOV and EFOV, respectively. In contrast, axial FWHMs increased with the MRD and ranged between 1.1 and 2.0 mm in the CFOV and between 1.5 and 2.7 mm in the EFOV for a MRD between 1 and 79. AFORE improved the axial resolution for a large part of the FOV, but image noise increased. OSEM3D/MAP yielded uniform spatial resolution in all directions, with an average FWHM of 1.65 ± 0.06 mm. Sensitivity in the CFOV for the default energy and coincidence time window was 0.068; peak sensitivity was 0.111. Conclusion: The Inveon showed high spatial resolution and high sensitivity, both of which can be maintained using OSEM3D/MAP reconstruction instead of rebinning and 2D algorithms.

Role of complement activation in hypersensitivity reactions to doxil and hynic PEG liposomes: experimental and clinical studies.

ABSTRACT Pegylated liposomal doxorubicin (Doxil) and 99mTc-HYNIC PEG liposomes (HPL) were reported earlier to cause hypersensitivity reactions (HSRs) in a substantial percentage of patients treated i.v. with these formulations. Here we report that (1) Doxil, HPL, pegylated phosphatidylethanolamine (PEG-PE)-containing empty liposomes matched with Doxil and HPL in size and lipid composition, and phosphatidylglycerol (PG)-containing negatively charged vesicles were potent C activators in human serum in vitro, whereas small neutral liposomes caused no C activation. (2) Doxil and other size-matched PEG-PE and/or PG-containing liposomes also caused massive cardiopulmonary distress with anaphylactoid shock in pigs via C activation, whereas equivalent neutral liposomes caused no hemodynamic changes. (3) A clinical study showed more frequent and greater C activation in patients displaying HSR than in non-reactive patients. These data suggest that liposome-induced HSRs in susceptible individuals may be due to C activation, which, in turn, is due to the presence of negatively charged PEG-PE in these vesicles.

A novel facile method of labeling octreotide with (18)F-fluorine.

Several methods have been developed to label peptides with 18F. However, in general these are laborious and require a multistep synthesis. We present a facile method based on the chelation of 18F-aluminum fluoride (Al18F) by 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). The method is characterized by the labeling of NOTA-octreotide (NOTA-d-Phe-cyclo[Cys-Phe-d-Trp-Lys-Thr-Cys]-Throl (MH+ 1305) [IMP466]) with 18F. Methods: Octreotide was conjugated with the NOTA chelate and labeled with 18F in a 2-step, 1-pot method. The labeling procedure was optimized with regard to the labeling buffer, peptide, and aluminum concentration. Radiochemical yield, specific activity, in vitro stability, and receptor affinity were determined. Biodistribution of 18F-IMP466 was studied in AR42J tumor–bearing mice and compared with that of 68Ga-labeled IMP466. In addition, small-animal PET/CT images were acquired. Results: IMP466 was labeled with Al18F in a single step with 50% yield. The labeled product was purified by high-performance liquid chromatography to remove unbound Al18F and unlabeled peptide. The radiolabeling, including purification, was performed in 45 min. The specific activity was 45,000 GBq/mmol, and the peptide was stable in serum for 4 h at 37°C. Labeling was performed at pH 4.1 in sodium citrate, sodium acetate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 2-(N-morpholino)ethanesulfonic acid buffer and was optimal in sodium acetate buffer. The apparent 50% inhibitory concentration of the 19F-labeled IMP466 determined on AR42J cells was 3.6 nM. Biodistribution studies at 2 h after injection showed a high tumor uptake of 18F-IMP466 (28.3 ± 5.2 percentage injected dose per gram [%ID/g]; tumor-to-blood ratio, 300 ± 90), which could be blocked by an excess of unlabeled peptide (8.6 ± 0.7 %ID/g), indicating that the accumulation in the tumor was receptor-mediated. Biodistribution of 68Ga-IMP466 was similar to that of 18F-IMP466. 18F-IMP466 was stable in vivo, because bone uptake was only 0.4 ± 0.2 %ID/g, whereas free Al18F accumulated rapidly in the bone (36.9 ± 5.0 %ID/g at 2 h after injection). Small-animal PET/CT scans showed excellent tumor delineation and high preferential accumulation in the tumor. Conclusion: NOTA-octreotide could be labeled rapidly and efficiently with 18F using a 2-step, 1-pot method. The compound was stable in vivo and showed rapid accretion in somatostatin receptor subtype 2–expressing AR42J tumors in nude mice. This method can be used to label other NOTA-conjugated compounds with 18F.

Image-quality assessment for several positron emitters using the NEMA NU 4-2008 standards in the Siemens Inveon small-animal PET scanner.

The positron emitters 18F, 68Ga, 124I, and 89Zr are all relevant in small-animal PET. Each of these radionuclides has different positron energies and ranges and a different fraction of single photons emitted. Average positron ranges larger than the intrinsic spatial resolution of the scanner (for 124I and 68Ga) will deteriorate the effective spatial resolution and activity recovery coefficient (RC) for small lesions or phantom structures. The presence of single photons (for 124I and 89Zr) could increase image noise and spillover ratios (SORs). Methods: Image noise, expressed as percentage SD in a uniform region (%SD), RC, and SOR (in air and water) were determined using the NEMA NU 4 small-animal image-quality phantom filled with 3.7 MBq of total activity of 18F, 68Ga, 124I, or 89Zr. Filtered backprojection (FBP), ordered-subset expectation maximization in 2 dimensions, and maximum a posteriori (MAP) reconstructions were compared. In addition to the NEMA NU 4 image-quality parameters, spatial resolutions were determined using small glass capillaries filled with these radionuclides in a water environment. Results: The %SD for 18F, 68Ga, 124I, and 89Zr using FBP was 6.27, 6.40, 6.74, and 5.83, respectively. The respective RCs were 0.21, 0.11, 0.12, and 0.19 for the 1-mm-diameter rod and 0.97, 0.65, 0.64, and 0.88 for the 5-mm-diameter rod. SORs in air were 0.01, 0.03, 0.04, and 0.01, respectively, and in water 0.02, 0.10, 0.13, and 0.02. Other reconstruction algorithms gave similar differences between the radionuclides. MAP produced the highest RCs. For the glass capillaries using FBP, the full widths at half maximum for 18F, 68Ga, 124I, and 89Zr were 1.81, 2.46, 2.38, and 1.99 mm, respectively. The corresponding full widths at tenth maximum were 3.57, 6.52, 5.87, and 4.01 mm. Conclusion: With the intrinsic spatial resolution (≈1.5 mm) of this latest-generation small-animal PET scanner, the finite positron range has become the limiting factor for the overall spatial resolution and activity recovery in small structures imaged with 124I and 68Ga. The presence of single photons had only a limited effect on the image noise. MAP, as compared with the other reconstruction algorithms, increased RC and decreased %SD and SOR.

A new technique for in vivo imaging of specific GLP-1 binding sites: First results in small rodents

EXPERIMENTAL OBJECTIVES In vivo imaging of GLP-1 receptor-positive tissues may allow examination of physiologic and pathophysiologic processes. Based on the GLP-1 analog Exendin 4, we have developed a radiolabeled compound specifically targeting the GLP-1 receptor (DTPA-Lys40-Exendin 4). This work aims to detect GLP-1 receptor-positive tissues by biodistribution studies and in vivo small animal imaging studies. For in vivo imaging, a high-resolution multi-pinhole SPECT (single photon emission computed tomography) system was used in conjunction with an MRI (magnetic resonance imaging) system for image fusion. RESULTS DTPA-Lys40-Exendin 4 can be labeled with 111In to high specific activity (40 GBq/micromol). The radiochemical purity reliably exceeded 95%. Using this compound for in vivo small animal imaging of rats and mice as well as for biodistribution studies, specific GLP-1 binding sites could be detected in stomach, pancreas, lung, adrenals, and pituitary. Receptor-positive tissues were visualized with a high-resolution SPECT system with a resolution of less than 1 mm. CONCLUSIONS The new technique using DTPA-Lys40-Exendin 4 allows highly sensitive imaging of GLP-1 receptor-positive tissues in vivo. Therefore, intra-individual follow-up studies of GLP-1 receptor-positive tissue could be conducted in vivo.

Pretargeted Immuno–Positron Emission Tomography Imaging of Carcinoembryonic Antigen–Expressing Tumors with a Bispecific Antibody and a 68Ga- and 18F-Labeled Hapten Peptide in Mice with Human Tumor Xenografts

18F-Fluorodeoxyglucose (18F-FDG) is the most common molecular imaging agent in oncology, with a high sensitivity and specificity for detecting several cancers. Antibodies could enhance specificity; therefore, procedures were developed for radiolabeling a small (∼1451 Da) hapten peptide with 68Ga or 18F to compare their specificity with 18F-FDG for detecting tumors using a pretargeting procedure. Mice were implanted with carcinoembryonic antigen (CEA; CEACAM5)–expressing LS174T human colonic tumors and a CEA-negative tumor, or an inflammation was induced in thigh muscle. A bispecific monoclonal anti-CEA × anti-hapten antibody was given to mice, and 16 hours later, 5 MBq of 68Ga- or 18F-labeled hapten peptides were administered intravenously. Within 1 hour, tissues showed high and specific targeting of 68Ga-IMP-288, with 10.7 ± 3.6% ID/g uptake in the tumor and very low uptake in normal tissues (e.g., tumor-to-blood ratio of 69.9 ± 32.3), in a CEA-negative tumor (0.35 ± 0.35% ID/g), and inflamed muscle (0.72 ± 0.20% ID/g). 18F-FDG localized efficiently in the tumor (7.42 ± 0.20% ID/g) but also in the inflamed muscle (4.07 ± 1.13% ID/g) and in several normal tissues; thus, pretargeted 68Ga-IMP-288 provided better specificity and sensitivity. Positron emission tomography (PET)/computed tomography images reinforced the improved specificity of the pretargeting method. 18F-labeled IMP-449 distributed similarly in the tumor and normal tissues as the 68Ga-labeled IMP-288, indicating that either radiolabeled hapten peptide could be used. Thus, pretargeted immuno-PET does exceptionally well with short-lived radionuclides and is a highly sensitive procedure that is more specific than 18F-FDG-PET. Mol Cancer Ther; 9(4); 1019–27. ©2010 AACR.

Correlation of [18F]FMISO autoradiography and pimonodazole immunohistochemistry in human head and neck carcinoma xenografts.

PurposeTumour cell hypoxia is a common feature in solid tumours adversely affecting radiosensitivity and chemosensitivity in head and neck squamous cell carcinomas. Positron emission tomography (PET) using the tracer [18F]fluoromisonidazole ([18F]FMISO) is most frequently used for non-invasive evaluation of hypoxia in human tumours. A series of ten human head and neck xenograft tumour lines was used to validate [18F]FMISO as hypoxia marker at the microregional level.MethodsAutoradiography after injection of [18F]FMISO was compared with immunohistochemical staining for the hypoxic cell marker pimonidazole in the same tumour sections of ten different human head and neck xenograft tumour lines. The methods were compared: first, qualitatively considering the microarchitecture; second, by obtaining a pixel-by-pixel correlation of both markers at the microregional level; third, by measuring the signal intensity of both images; and fourth, by calculating the hypoxic fractions by pimonidazole labelling.ResultsThe pattern of [18F]FMISO signal was dependent on the distribution of hypoxia at the microregional level. The comparison of [18F]FMISO autoradiography and pimonidazole immunohistochemistry by pixel-by-pixel analysis revealed moderate correlations. In five tumour lines, a significant correlation between the mean [18F]FMISO and pimonidazole signal intensity was found (range, r2 = 0.91 to r2 = 0.99). Comparison of the tumour lines with respect to the microregional distribution pattern of hypoxia revealed that the correlation between the mean signal intensities strongly depended on the microarchitecture. Overall, a weak but significant correlation between hypoxic fractions based on pimonidazole labeling and the mean [18F]FMISO signal intensity was observed (r2 = 0.18, p = 0.02). For the three tumour models with a ribbon-like microregional distribution pattern of hypoxia, the correlation between the hypoxic fraction and the mean [18F]FMISO signal intensity was much stronger and more significant (r2 = 0.73, p < 0.001) than for the tumours with a more homogenous, patchy, microregional distribution pattern of hypoxia.ConclusionDifferent patterns of [18F]FMISO accumulation dependent on the underlying microregional distribution of hypoxia were found in ten head and neck xenograft tumours. A weak albeit significant correlation was found between the mean [18F]FMISO signal intensity and the hypoxic fraction of the tumours. In larger clinical tumours, [18F]FMISO–PET provides information on the tumour oxygenation status on a global level, facilitating dose painting in radiation treatment planning. However, caution must be taken when studying small tumour subvolumes as accumulation of the tracer depends on the presence of hypoxia and on the tumour microarchitecture.

Application of Metal‐Free Triazole Formation in the Synthesis of Cyclic RGD–DTPA Conjugates

The tandem 1,3‐dipolar cycloaddition‐retro‐Diels–Alder (tandem crDA) reaction is presented as a versatile method for metal‐free chemoselective conjugation of a DTPA radiolabel to N‐δ‐azido‐cyclo(‐Arg‐Gly‐Asp‐d‐Phe‐Orn‐) via oxanorbornadiene derivatives. To this end, the behavior of several trifluoromethyl‐substituted oxanorbornadiene derivatives in the 1,3‐dipolar cycloaddition was studied and optimized to give a clean and efficient method for bio‐orthogonal ligation in an aqueous environment. After radioisotope treatment, the resulting 111In‐labeled c(RGD)‐CF3‐triazole‐DTPA conjugate was subjected to preliminary biological evaluation and showed high affinity for αvβ3 (IC50=192 nM) and favorable pharmacokinetics.