Site-Specific Labeling of scVEGF with Fluorine-18 for Positron Emission Tomography Imaging

Vascular endothelial growth factor (VEGF) is one of the most important mediators of angiogenesis. Single-chain (sc)-VEGF protein containing an N-terminal Cys-tag has been designed for site-specific modification with a variety of imaging and therapeutic moieties. Site-specific labeling of scVEGF with thiol-reactive prosthetic group, N-[2-(4-18F-fluorobenzamido) ethyl] maleimide ([18F]FBEM) for positron emission tomography (PET) imaging of VEFGR may provide a new tracer which has great potential for clinical translation

Quantitative Analysis and Comparison Study of [18F]AlF-NOTA-PRGD2, [18F]FPPRGD2 and [68Ga]Ga-NOTA-PRGD2 Using a Reference Tissue Model

With favorable pharmacokinetics and binding affinity for αvβ3 integrin, 18F-labeled dimeric cyclic RGD peptide ([18F]FPPRGD2) has been intensively used as a PET imaging probe for lesion detection and therapy response monitoring. A recently introduced kit formulation method, which uses an 18F-fluoride-aluminum complex labeled RGD tracer ([18F]AlF-NOTA-PRGD2), provides a strategy for simplifying the labeling procedure to facilitate clinical translation. Meanwhile, an easy-to-prepare 68Ga-labeled NOTA-PRGD2 has also been reported to have promising properties for imaging integrin αvβ3. The purpose of this study is to quantitatively compare the pharmacokinetic parameters of [18F]FPPRGD2, [18F]AlF-NOTA-PRGD2, and [68Ga]Ga-NOTA-PRGD2. U87MG tumor-bearing mice underwent 60-min dynamic PET scans following the injection of three tracers. Kinetic parameters were calculated using Logan graphical analysis with reference tissue. Parametric maps were generated using voxel-level modeling. All three compounds showed high binding potential (BpND = k3/k4) in tumor voxels. [18F]AlF-NOTA-PRGD2 showed comparable BpND value (3.75±0.65) with those of [18F]FPPRGD2 (3.39±0.84) and [68Ga]Ga-NOTA-PRGD2 (3.09±0.21) (p>0.05). Little difference was found in volume of distribution (VT) among these three RGD tracers in tumor, liver and muscle. Parametric maps showed similar kinetic parameters for all three tracers. We also demonstrated that the impact of non-specific binding could be eliminated in the kinetic analysis. Consequently, kinetic parameter estimation showed more comparable results among groups than static image analysis. In conclusion, [18F]AlF-NOTA-PRGD2 and [68Ga]Ga-NOTA-PRGD2 have comparable pharmacokinetics and quantitative parameters compared to those of [18F]FPPRGD2. Despite the apparent difference in tumor uptake (%ID/g determined from static images) and clearance pattern, the actual specific binding component extrapolated from kinetic modeling appears to be comparable for all three dimeric RGD tracers

pH-Sensitive Fluorescent Dyes: Are They Really pH-Sensitive in Cells?

National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health; NIBIB; National Institute of Standards and TechnologyChemically synthesized near-infrared aza-BODIPY dyes displayed off-on fluorescence at acidic pH (pK(a) = 6.2-6.6) through the suppression of the photoinduced electron transfer and/or internal charge transfer process. The apparent pK(a)s of the dyes were shifted well above physiological pH in a hydrophobic microenvironment, which led to "turned-on" fluorescence in micelles and liposomes at neutral and basic pH. Bovine serum albumin also activated the fluorescence, though to a much lesser extent. When these small molecular dyes entered cells, instead of being fluorescent only in acidic organelles, the whole cytoplasm exhibited fluorescence, with a signal/background ratio as high as similar to 10 in no-wash live-cell imaging. The dye 1-labeled cells remained highly fluorescent even after 3 days. Moreover, slight variations of the dye structure resulted in significantly different intracellular fluorescence behaviors, possibly because of their different cellular uptake and intracellular activation capabilities. After the separation of cellular components, the fraction of plasma membrane and endoplasmic reticulum showed the highest fluorescence, further confirming the fluorescence activation by membrane structures. The fluorescence intensity of these dyes at different intracellular pHs (6.80 and 8.00) did not differ significantly, indicating that intracellular pH did not play a critical role. Altogether, we showed here for the first time that the fluorescence of pH-sensitive aza-BODIPY dyes was switched intracellularly not by acidic pH, but by intracellular membranes (and proteins as well). The excellent membrane permeability, ultrahigh fluorescence contrast ratio, persistent fluorescent signal, and minimal biological interference of dye 1 make it an ideal choice for live-cell imaging and in vivo cell tracking. These findings also imply that the intracellular fluorescence properties of pH-sensitive dyes should be carefully examined before they are used as pH indicators

Evaluation of an [18F]AlF-NOTA Analog of Exendin-4 for Imaging of GLP-1 Receptor in Insulinoma

<p><b>Introduction:</b> The GLP-1 receptor plays an important role in glucose homeostasis and thus is a very important target for diabetes therapy. The receptor is also overexpressed in insulinoma, a tumor of pancreatic beta-cells. We previously evaluated two fluorine-18-labeled analogs of exendin-4 prepared by conjugation with [¹⁸F]FBEM (N-[2-(4-[¹⁸F]fluorobenzamide)ethyl]maleimide). Both compounds demonstrated good tumor uptake, but the synthesis of the radiotracers was time consuming. To overcome this challenge, we developed a NOTA analog and performed radiolabeling using aluminum [¹⁸F]fluoride complexation.</p><p><b>Methods:</b> Cys⁴⁰-exendin-4 was conjugated with NOTA mono N-ethylmaleimide. [¹⁸F]AlF conjugation was conducted and the radiolabeled product purified by preparative HPLC. Dynamic and static PET imaging scans were conducted on nude mice with established INS-1 xenografts. Uptake of tumor and other major organs in static images was quantitated (%ID/g) and comparison with blocking studies was made. PET quantification was also compared with ex vivo biodistribution results.</p><p><b>Results:</b> The radiosynthesis provided [¹⁸F]AlF-NOTA-MAL-cys⁴⁰-exendin-4 in 23.6 &#177; 2.4 % radiochemical yield (uncorrected, n = 3) after HPLC; the process required about 55 min. The specific activity at time of injection ranged from 19.6 to 31.4 GBq (0.53-0.85 Ci)/&#181;mol. Tumor uptake had reached its maximum (16.09 &#177; 1.18% ID/g, n = 4) by 5 min and remained nearly constant for the duration of the study. Kidney uptake continued to increase throughout the entire one hour time course. Pre-injection of exendin-4 caused a marked reduction in tissue uptake with the major exception of liver and kidneys, in which uptake was not affected. HPLC analysis of the radioactive components in extracts of the tumor and plasma showed primarily parent compound at 60 min post-injection, whereas extracts of kidney and urine contained exclusively one polar radioactive component.</p><p><b>Conclusion:</b> The radiotracer is prepared in a simple one-step procedure and obtained in high specific activity after HPLC purification. [¹⁸F]AlF-NOTA-MAL-exendin-4 shows high tumor uptake and highly selective GLP-1 tissue uptake (INS-1 tumor, lung, pancreas), but still suffers from high kidney uptake.</p

PET imaging of CXCR4 using copper-64 labeled peptide antagonist

<p>Expression of CXCR4 in cancer has been found to correlate with poor prognosis and resistance to chemotherapy. In this study we developed a derivative of the CXCR4 peptide antagonist, T140-2D, that can be labeled easily with the PET isotope copper-64, and thereby enable <i>in vivo</i> visualization of CXCR4 in tumors. T140 was conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono (<i>N</i>-hydroxysuccinimide ester) (DOTA-NHS) to give T140-2D, which contains a DOTA molecule on each of the two lysine residues. ⁶⁴Cu-T140-2D was evaluated <i>in vitro</i> by migration and binding experiments, and <i>in vivo</i> by microPET imaging and biodistribution, in mice bearing CXCR4-positive and CXCR4-negative tumor xenografts. T140-2D was labeled with copper-64 to give ⁶⁴Cu-T140-2D in a high radiochemical yield of 86 &#177; 3% (not decay-corrected) and a specific activity of 0.28 - 0.30 mCi/&#181;g (10.36 - 11.1 MBq/&#181;g). ⁶⁴Cu-T140-2D had antagonistic and binding characteristics to CXCR4 that were similar to those of T140. <i>In vivo</i>, ⁶⁴Cu-T140-2D tended to bind to red blood cells and had to be used in a low specific activity form. In this new form ⁶⁴Cu-T140-2D enabled specific imaging of CXCR4-positive, but not CXCR4-negative tumors. Undesirably, however, ⁶⁴Cu-T140-2D also displayed high accumulation in the liver and kidneys. In conclusion, ⁶⁴Cu-T140-2D was easily labeled and, in its low activity form, enabled imaging of CXCR4 in tumors. It had high uptake, however, in metabolic organs. Further research with imaging tracers targeting CXCR4 is required.</p