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What is the optimal label to trace your compound?

Maarten Brom, PhD

With molecular imaging you can visualize the distribution of a drug in vivo. To trace your drug in the (human) body, you need to label the drug. You can label a drug with a wide variety of labels. Frequently used labels for in vivo imaging are radionuclide and fluorescent dyes. The choice for a fluorescent label or radionuclide depends on the purpose of your (clinical) study.

The difference between the imaging of radionuclides and fluorescent dyes.

Radionuclides and fluorescent dyes have different physical properties. The label choice depends on the overall aim of your study. As radionuclide imaging (PET and SPECT) has a good tissue penetration of gamma photons you can perform whole body imaging. Also, deeper seated organs or lesions can easily be detected with radionuclide imaging. Other advantages of radionuclide imaging are accurate quantification of radiotracer concentration, the possibility of repeated scanning and a high temporal resolution.

With fluorescence imaging only superficial lesions can be imaged. This is caused by the limited penetration depth of (fluorescent) light in tissue. Nevertheless, fluorescence imaging is characterized by a high spatial resolution. This makes it ideal to visualize the distribution of your fluorescently labeled drug at a microscopic level.

How to select the optimal radionuclide for your compound?

When choosing a radionuclide for your study, you still need to pick the optimal radionuclide to label your compound. The choice for the radionuclide depends on the chemical and biological characteristics of your compound. For example, the circulation time of a compound in the body determines the selection of the half-life of the radionuclide. Long circulating compounds in the body, such as antibodies, require a radionuclide with a long half-life. You can use a radionuclide with a short half-life for fast clearing compounds, such as small peptides.

The influence of labeling method on radionuclide selection

The labeling method can also influence the selection of the radionuclide. For example, you can label radiometals (e.g., gallium-68, indium-111 and zirconium-89) in a two-step approach (indirect labeling). First, you couple a chelator to your compound. After coupling of the chelator, you add the radiometal. Now, the chelator will form a complex with the radionuclide resulting in a radiolabeled compound. This strategy is mainly used for larger molecules (antibodies) and peptides. Since these molecules have a larger molecular weight the influence of the chelator of a considerable size has only a limited influence on the biological characteristics of your labeled compound.

If you develop small molecules, the size of the chelator is usually too large and is likely to influence the in vivo behavior of your compounds. Therefore, for the labeling of small molecules, a direct labeling method should be selected. With this method the radionuclide is directly labeled to or incorporated into the molecule of interest. Examples of radionuclides suitable for direct labeling are carbon-11, fluorine-18 and iodine-123. An example of a labeled small molecules is a glucose analog labeled with fluorine-18 ([18F]FDG) for the measurement of (tumor) glucose metabolism. Another example is the measurement of dopamine transporters in the brain by Ioflupane labeled with iodine-123 for the detection of Parkinson’s disease.

The influence of imaging modality on radionuclide selection

The imaging modality you choose also determines your choice for the radionuclide. Where SPECT-imaging is designed to detect gamma emitters, positron emitters must be selected for PET-imaging. Clinical PET has the advantage over SPECT in terms of improved resolution, sensitivity, and more accurate quantification. On the other hand, radionuclides and radiopharmaceuticals for SPECT are widely commercially available, where most of the PET radiopharmaceuticals require the presence of a cyclotron and a sophisticated GMP facility within the institution or a nearby institution. Due to the short half-life of most PET radionuclides, transportation over long distances is not feasible.

How to select the optimal fluorescent dye for your compound?

After excitation, a fluorescent dye emits light. Various fluorescent dyes are available in a range of wavelengths for excitation and emission. For in vivo application the excitation and emission wavelength are of importance for the selection of the fluorescent dye. At lower wavelengths, (visible light), the amount of absorption of light in tissue is high. This results in a lower penetration depth of the light, which makes it more difficult to detect deeper seated lesions. Thus, for in vivo application you can better use fluorescent dyes in the Near Infrared (NIR) spectrum (>700nm), as the penetration depth is higher due to lesser absorption by (oxy-)hemoglobin. Moreover, a lower background signal is observed due to lower autofluorescence. In the human body, several molecules are present that are fluorescent by itself. The excitation and emission spectra of these molecules are usually lower than the NIR spectrum, with lower autofluorescence as a result.

Currently, mostly fluorescent labeled antibodies are used in fluorescence imaging clinical trials. The labeling of your large antibodies with a relatively small fluorescent dye is not likely to change the biodistribution or bindings specificity of your antibody. However, when you use smaller compound such as peptides, the labeling with a fluorescent dye might influence the biodistribution and target-binding specificity. Moreover, many peptides are water soluble (hydrophilic). As a result, they are cleared via the kidneys in the human body. The labeled peptide is then excreted via the urine. Nevertheless, many fluorescent dyes are not water soluble (hydrophobic). These hydrophobic compounds are cleared via the liver into the bile and the metabolites will be excreted via the intestinal tract. This might result in a background signal from the liver and intestines, which cover the complete abdomen. When you label a peptide with a hydrophobic dye, this might change the biodistribution and should be considered during your selection of the dye.

Wondering what the optimal label for your compound and research goal is? At TRACER we have longstanding experience in labeling various compounds with a large range of radionuclides and fluorescent dyes.

We can assist you in the selection of the optimal label for your purpose. Contact us here.

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