Lieke Joosten

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.

Obstacles on the way to the clinical visualisation of beta cells: looking for the Aeneas of molecular imaging to navigate between Scylla and Charybdis.

For more than a decade, researchers have been trying to develop non-invasive imaging techniques for the in vivo measurement of viable pancreatic beta cells. However, in spite of intense research efforts, only one tracer for positron emission tomography (PET) imaging is currently under clinical evaluation. To many diabetologists it may remain unclear why the imaging world struggles to develop an effective method for non-invasive beta cell imaging (BCI), which could be useful for both research and clinical purposes. Here, we provide a concise overview of the obstacles and challenges encountered on the way to such BCI, in both native and transplanted islets. We discuss the major difficulties posed by the anatomical and cell biological features of pancreatic islets, as well as the chemical and physical limits of the main imaging modalities, with special focus on PET, SPECT and MRI. We conclude by indicating new avenues for future research in the field, based on several remarkable recent results.

PET and SPECT imaging of a radiolabeled minigastrin analogue conjugated with DOTA, NOTA, and NODAGA and labeled with (64)Cu, (68)Ga, and (111)In.

Cholecystokinin-2 (CCK-2) receptors, overexpressed in cancer types such as small cell lung cancers (SCLC) and medullary thyroid carcinomas (MTC), may serve as targets for peptide receptor radionuclide imaging. A variety of CCK and gastrin analogues has been developed, but a major drawback is metabolic instability or high kidney uptake. The minigastrin analogue PP-F11 has previously been shown to be a promising peptide for imaging of CCK-2 receptor positive tumors and was therefore further evaluated. The peptide was conjugated with one of the macrocyclic chelators DOTA, NOTA, or NODAGA. The peptide conjugates were then radiolabeled with either (68)Ga, (64)Cu, or (111)In. All (radio)labeled compounds were evaluated in vitro (IC50) and in vivo (biodistribution and PET/CT and SPECT/CT imaging). IC50 values were in the low nanomolar range for all compounds (0.79-1.51 nM). In the biodistribution studies, (68)Ga- and (111)In-labeled peptides showed higher tumor-to-background ratios than the (64)Cu-labeled compounds. All tested radiolabeled compounds clearly visualized the CCK2 receptor positive tumor in PET or SPECT imaging. The chelator did not seem to affect in vivo behavior of the peptide for (111)In- and (68)Ga-labeled peptides. In contrast, the biodistribution of the (64)Cu-labeled peptides showed high uptake in the liver and in other organs, most likely caused by high blood levels, probably due to dissociation of (64)Cu from the chelator and subsequent transchelation to proteins. Based on the present study, (68)Ga-DOTA-PP-F11 might be a promising radiopharmaceutical for PET/CT imaging of CCK2 receptor expressing tumors such as MTC and SCLC. Clinical studies are warranted to investigate the potential of this tracer.

Radiolabelled GLP-1 analogues for in vivo targeting of insulinomas.

Internalizing agonists are usually selected for peptide receptor targeting. There is increasing evidence that non-internalizing receptor antagonists can be used for this purpose. We investigated whether the glucagon-like peptide-1 receptor (GLP-1R) antagonist exendin(9-39) can be used for in vivo targeting of GLP-1R expressing tumours and compared the in vitro and in vivo characteristics with the GLP-1R agonists exendin-3 and exendin-4. The binding and internalization kinetics of labelled [Lys(40) (DTPA)]exendin-3, [Lys(40) (DTPA)]exendin-4 and [Lys(40) (DTPA)]exendin(9-39) were determined in vitro using INS-1 cells. The in vivo targeting properties of [Lys(40) ((111) In-DTPA)]exendin-3, [Lys(40) ((111) In-DTPA)]exendin-4 and [Lys(40) ((111) In-DTPA)]exendin(9-39) were examined in BALB/c nude mice with subcutaneous INS-1 tumours. (nat) In-labelled [Lys(40) (DTPA)]exendin-3, [Lys(40) (DTPA)]exendin-4 and [Lys(40) (DTPA)]exendin(9-39) exhibited similar IC(50) values (13.5, 14.4 and 13.4 n m, respectively) and bound to 26 × 10(3) , 41 × 10(3) and 37 × 10(3) receptors per cell, respectively. [Lys(40) ((111) In-DTPA)]exendin-3 and [Lys(40) ((111) In-DTPA)]exendin-4 showed rapid in vitro binding and internalization kinetics, whereas [Lys(40) ((111) In-DTPA)]exendin(9-39) showed lower binding and minimal internalization in vitro. In mice, high specific uptake of [Lys(40) ((111) In-DTPA)]exendin-3 [25.0 ± 6.0% injected dose (ID) g(-1) ] in the tumour was observed at 0.5 h post-injection (p.i.) with similar uptake up to 4 h p.i. [Lys(40) ((111) In-DTPA)]exendin-4 showed higher tumour uptake at 1 and 4 h p.i. (40.8 ± 7.0 and 41.9 ± 7.2% ID g(-1), respectively). Remarkably, [Lys(40) ((111) In-DTPA)]exendin(9-39) showed only low specific uptake in the tumour at 0.5 h p.i. (3.2 ± 0.7% ID g(-1)), rapidly decreasing over time. In conclusion, the GLP-1R agonists [Lys(40) (DTPA)]exendin-3 and [Lys(40) (DTPA)]exendin-4 labelled with (111) In could be useful for in vivo GLP-1R targeting, whereas [Lys(40) (DTPA)]exendin(9-39) is not suited for in vivo targeting of the GLP-1R.

Improved labelling of DTPA- and DOTA-conjugated peptides and antibodies with 111In in HEPES and MES buffer

BackgroundIn single photon emission computed tomography [SPECT], high specific activity of 111In-labelled tracers will allow administration of low amounts of tracer to prevent receptor saturation and/or side effects. To increase the specific activity, we studied the effect of the buffer used during the labelling procedure: NaAc, NH4Ac, HEPES and MES buffer. The effect of the ageing of the 111InCl3 stock and cadmium contamination, the decay product of 111In, was also examined in these buffers.MethodsEscalating amounts of 111InCl3 were added to 1 μg of the diethylene triamine pentaacetic acid [DTPA]- and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid [DOTA]-conjugated compounds (exendin-3, octreotide and anti-carbonic anhydrase IX [CAIX] antibody). Five volumes of 2-(N-morpholino)ethanesulfonic acid [MES], 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], NH4Ac or NaAc (0.1 M, pH 5.5) were added. After 20 min at 20°C (DTPA-conjugated compounds), at 95°C (DOTA-exendin-3 and DOTA-octreotide) or at 45°C (DOTA-anti-CAIX antibody), the labelling efficiency was determined by instant thin layer chromatography. The effect of the ageing of the 111InCl3 stock on the labelling efficiency of DTPA-exendin-3 as well as the effect of increasing concentrations of Cd2+ (the decay product of 111In) were also examined.ResultsSpecific activities obtained for DTPA-octreotide and DOTA-anti-CAIX antibody were five times higher in MES and HEPES buffer. Radiolabelling of DTPA-exendin-3, DOTA-exendin-3 and DTPA-anti-CAIX antibody in MES and HEPES buffer resulted in twofold higher specific activities than that in NaAc and NH4Ac. Labelling of DTPA-exendin-3 decreased with 66% and 73% for NaAc and NH4Ac, respectively, at day 11 after the production date of 111InCl3, while for MES and HEPES, the maximal decrease in the specific activity was 10% and 4% at day 11, respectively. The presence of 1 pM Cd2+ in the labelling mixture of DTPA-exendin-3 in NaAc and NH4Ac markedly reduced the labelling efficiency, whereas Cd2+ concentrations up to 0.1 nM did not affect the labelling efficiency in MES and HEPES buffer.ConclusionsWe showed improved labelling of DTPA- and DOTA-conjugated compounds with 111In in HEPES and MES buffer. The enhanced labelling efficiency appears to be due to the reduced competitive chelation of cadmium. The enhanced labelling efficiency will allow more sensitive imaging of the biomarkers with SPECT.

Preclinical evaluation of 68Ga-DOTA-minigastrin for the detection of cholecystokinin-2/gastrin receptor-positive tumors

In comparison to somatostatin receptor scintigraphy, gastrin receptor scintigraphy using 111In-DTPA-minigastrin (MG0) showed added value in diagnosing neuroendocrine tumors. We investigated whether the 68Ga-labeled gastrin analogue DOTA-MG0 is suited for positron emission tomography (PET), which could improve image quality. Targeting of cholecystokinin-2 (CCK2)/gastrin receptor-positive tumor cells with DOTA-MG0 labeled with either 111In or 68Ga in vitro was investigated using the AR42J rat tumor cell line. Biodistribution was examined in BALB/c nude mice with a subcutaneous AR42J tumor. In vivo PET imaging was performed using a preclinical PET-computed tomographic scanner. DOTA-MG0 showed high receptor affinity in vitro. Biodistribution studies revealed high tumor uptake of 68Ga-DOTA-MG0: 4.4 ± 1.3 %ID/g at 1 hour postinjection. Coadministration of an excess unlabeled peptide blocked the tumor uptake (0.7 ± 0.1 %ID/g), indicating CCK2/gastrin receptor-mediated uptake (p = .0005). The biodistribution of 68Ga-DOTA-MG0 was similar to that of 111In-DOTA-MG0. Subcutaneous and intraperitoneal tumors were clearly visualized by small-animal PET imaging with 5 MBq 68Ga-DOTA-MG0. 111In- and 68Ga-labeled DOTA-MG0 specifically accumulate in CCK2/gastrin receptor-positive AR42J tumors with similar biodistribution apart from the kidneys. AR42J tumors were clearly visualized by microPET. Therefore, 68Ga-DOTA-MG0 is a promising tracer for PET imaging of CCK2/gastrin receptor-positive tumors in humans.

Combined Optical Coherence and Fluorescence Microscopy to assess dynamics and specificity of pancreatic beta-cell tracers

The identification of a beta-cell tracer is a major quest in diabetes research. However, since MRI, PET and SPECT cannot resolve individual islets, optical techniques are required to assess the specificity of these tracers. We propose to combine Optical Coherence Microscopy (OCM) with fluorescence detection in a single optical platform to facilitate these initial screening steps from cell culture up to living rodents. OCM can image islets and vascularization without any labeling. Thereby, it alleviates the need of both genetically modified mice to detect islets and injection of external dye to reveal vascularization. We characterized Cy5.5-exendin-3, an agonist of glucagon-like peptide 1 receptor (GLP1R), for which other imaging modalities have been used and can serve as a reference. Cultured cells transfected with GLP1R and incubated with Cy5.5-exendin-3 show full tracer internalization. We determined that a dose of 1 μg of Cy5.5-exendin-3 is sufficient to optically detect in vivo the tracer in islets with a high specificity. In a next step, time-lapse OCM imaging was used to monitor the rapid and specific tracer accumulation in murine islets and its persistence over hours. This optical platform represents a versatile toolbox for selecting beta-cell specific markers for diabetes research and future clinical diagnosis.

In Vitro and In Vivo Characterization of Three 68Ga- and 111In-Labeled Peptides for Cholecystokinin Receptor Imaging:

Cholecystokinin (CCK) receptors are overexpressed in several human tumor types, such as medullary thyroid carcinomas and small cell lung cancers. Several ligands for the CCK2 receptor (CCK2R) have been developed for radionuclide targeting of these tumors. In this study, we evaluated whether radiolabeled DOTA-sCCK8 and its stabilized derivative, DOTA-sCCK8[Phe2(p-CH2SO3H), Nle3,6], are suitable for imaging of CCK2R-positive tumors, using DOTA-MG0 as a reference. In vivo targeting of CCK2R-positive tumors with DOTA-sCCK8, DOTA-sCCK8[Phe2(p-CH2SO3H), Nle3,6], and DOTA-MG0, labeled with 111In or 68Ga, was evaluated in BALB/c nude mice with a subcutaneous A431-CCK2R tumor. Biodistribution studies and single-photon emission computed tomography (SPECT) and positron emission tomography (PET) were performed at 1 hour postinjection. All peptides specifically accreted in the CCK2R-expressing tumors. Both 111In-DOTA-sCCK8 and 111In-DOTA-sCCK8[Phe2(p-CH2SO3H), Nle3,6] showed good tumor retention (4.65% ID/g and 5.44% ID/g, respectively, at 4 hours postinjection). On PET/computed tomographic (CT) and SPECT/CT scans, subcutaneous A431-CCK2R tumors were clearly visualized with low uptake of sCCK8 peptides in the intestines. Whereas radiolabeled DOTA-MG0 showed high kidney uptake (70% ID/g), the sCCK8 peptides showed low uptake in the kidneys. Sulfated CCK8 analogues combined high tumor uptake with low retention in the kidney and are therefore promising tracers for imaging of CCK2R-positive tumors.

SPECT-OPT multimodal imaging enables accurate evaluation of radiotracers for beta-cell mass assessments

Single Photon Emission Computed Tomography (SPECT) has become a promising experimental approach to monitor changes in β-cell mass (BCM) during diabetes progression. SPECT imaging of pancreatic islets is most commonly cross-validated by stereological analysis of histological pancreatic sections after insulin staining. Typically, stereological methods do not accurately determine the total β-cell volume, which is inconvenient when correlating total pancreatic tracer uptake with BCM. Alternative methods are therefore warranted to cross-validate β-cell imaging using radiotracers. In this study, we introduce multimodal SPECT - optical projection tomography (OPT) imaging as an accurate approach to cross-validate radionuclide-based imaging of β-cells. Uptake of a promising radiotracer for β-cell imaging by SPECT, 111In-exendin-3, was measured by ex vivo-SPECT and cross evaluated by 3D quantitative OPT imaging as well as with histology within healthy and alloxan-treated Brown Norway rat pancreata. SPECT signal was in excellent linear correlation with OPT data as compared to histology. While histological determination of islet spatial distribution was challenging, SPECT and OPT revealed similar distribution patterns of 111In-exendin-3 and insulin positive β-cell volumes between different pancreatic lobes, both visually and quantitatively. We propose ex vivo SPECT-OPT multimodal imaging as a highly accurate strategy for validating the performance of β-cell radiotracers.

SPECT of Transplanted Islets of Langerhans by Dopamine 2 Receptor Targeting in a Rat Model.

Pancreatic islet transplantation can be a more permanent treatment for type 1 diabetes compared to daily insulin administration. Quantitative and longitudinal noninvasive imaging of viable transplanted islets might help to further improve this novel therapy. Since islets express dopamine 2 (D2) receptors, they could be visualized by targeting this receptor. Therefore, the D2 receptor antagonist based tracer [(125/123)I][IBZM] was selected to visualize transplanted islets in a rat model. BZM was radioiodinated, and the labeling was optimized for position 3 of the aromatic ring. [(125)I]-3-IBZM was characterized in vitro using INS-1 cells and isolated islets. Subsequently, 1,000 islets were transplanted in the calf muscle of WAG/Rij rats and SPECT/CT images were acquired 6 weeks after transplantation. Finally, the graft containing muscle was dissected and analyzed immunohistochemically. Oxidative radioiodination resulted in 3 IBZM isomers with different receptor affinities. The use of 0.6 mg/mL chloramine-T hydrate resulted in high yield formation of predominantly [(125)I]-3-IBZM, the isomer harboring the highest receptor affinity. The tracer showed D2 receptor mediated binding to isolated islets in vitro. The transplant could be visualized by SPECT 6 weeks after transplantation. The transplants could be localized in the calf muscle and showed insulin and glucagon expression, indicating targeting of viable and functional islets in the transplant. Radioiodination was optimized to produce high yields of [(125)I]-3-IBZM, the isomer showing optimal D2R binding. Furthermore, [(123)I]IBZM specifically targets the D2 receptors on transplanted islets. In conclusion, this tracer shows potential for noninvasive in vivo detection of islets grafted in the muscle by D2 receptor targeting.