Molecular Imaging Strategies Imaging Targets

Imaging approaches may target different aspects that relate to the efficacy of drugs: its biodistribution, the interaction of the drug with its therapeutic target, the initiation of the signaling cascade, or the response of the biological system in terms of morphological, physiological, or metabolic changes (Figure 7.3). In the following list, these different aspects will be addressed.

a. Drug biodistribution: Unfavorable PK properties are an important reason for failure of drugs during development. In view of this fact, detailed knowledge on the drug's biodistribution is of key importance in the development of novel therapeutics. During the preclinical phase this information is commonly obtained from quantitative whole-body autoradiographic studies, which measures the distribution of radiolabeled drug molecules. More recently, matrix-assisted laser desorption and ionization mass spectrometric (MALDI-MS) imaging has been introduced for PK studies in tissue samples. Molecular identification is based on the mass determination; hence, MALDI-MS imaging does not require labeling with radioisotopes. In addition, using the mass filter parent molecule and metabolites can be distinguished, in contrast to radiolabel-based techniques. Both autoradiographic and MALDI-MS imaging are ex vivo techniques and will not be discussed further.

In vivo drug biodistribution studies almost exclusively use PET. As drug labeling should not affect its PK and pharmacodynamic properties, its molecular structure must be unaffected by the introduction of a reporter group. The only possibility to achieve this is isotopic substitution by a radionuclide. Moreover, introduction of a radioactive

Ligand biodistribution

Receptor distribution and occupancy

Pathway activity

Extracellular

System response: morphological, physiological, metabolic cellular, and molecular readout

Extracellular

System response: morphological, physiological, metabolic cellular, and molecular readout

FIGURE 7.3 Imaging targets relevant for DDD. Currently available imaging techniques allow visualization and quantification of the drug's mechanism of action. Labeling of the drug molecule itself (or of a competitive receptor ligand) reveals information on its biodistribution and receptor interaction. The expression level of a receptor can be visualized using specific reporter ligands or following a reporter gene strategy. Activation of the signaling cascade is visualized by targeting individual pathway molecules (e.g., caspases for studying apoptosis) or by measuring protein-protein interaction (see text). Finally the result of the therapeutic intervention such as morphological, physiological, metabolic, cellular, or molecular changes can be monitored.

FIGURE 7.3 Imaging targets relevant for DDD. Currently available imaging techniques allow visualization and quantification of the drug's mechanism of action. Labeling of the drug molecule itself (or of a competitive receptor ligand) reveals information on its biodistribution and receptor interaction. The expression level of a receptor can be visualized using specific reporter ligands or following a reporter gene strategy. Activation of the signaling cascade is visualized by targeting individual pathway molecules (e.g., caspases for studying apoptosis) or by measuring protein-protein interaction (see text). Finally the result of the therapeutic intervention such as morphological, physiological, metabolic, cellular, or molecular changes can be monitored.

reporter nuclide yields the sensitivity that is required to detect small amounts of the drug ligand in tissue.

b. Expression of the molecular target: A critical step in early drug discovery is target validation, i.e., demonstration of the presence of a drug target in the tissue of interest. The common strategy to visualize and quantify the presence of a drug target, such as a membrane receptor or an enzyme, uses target-specific imaging probes, the vast majority using either radionuclides or fluorescent dyes as reporter moiety. There are numerous examples of such studies (Section 7.3.1). It is important to realize that unless one uses target-activatable probes, it cannot be discriminated whether the signal observed arises from the reporter fraction that is bound to its target or just from free or unspecifically bound molecules. Thus, it is important to wait until the unbound probe is cleared from circulation. Alternatively, reporter gene assays can be used to visualize target expression.

c. Imaging pathway activities: Two strategies can be pursued to study pathway activities, either by monitoring critical molecules in the signal transduction cascade or by visualizing protein-protein interactions. The first approach uses the concept outlined in the previous paragraph. For example, a reporter gene assay has been developed to visualize the activity of caspases-3, a critical player in cellular apoptosis. Signal propagation relies on protein-protein interactions. A number of assays have been developed to study these key processes in cellular systems; some of them have been translated for applications in intact animals such as the two-hybrid assay or the protein fragment complementation assay. As an example, a split luciferase assay has been developed to study the interaction of the two proteins FRB and FKBP12, which is induced by the administration of the macrolide rapamycin.

d. Monitoring cell migration: Monitoring the trafficking and fate of labeled cells in vivo has a wide range of applications in DDD. Tumor cells have been transfected to express fluorescent or bioluminescent proteins for the noninvasive assessment of tumor growth or metastasis load in murine tumor models. Inflammatory processes have been studied using MRI by monitoring the infiltration of monocytes and lymphocytes labeled with superparamagnetic iron oxide nanoparticles into inflamed tissue. Cell therapy is becoming an increasingly important therapeutic strategy requiring tools to visualize the location, migration, and viability of stem or progenitor cells. The fate of such cells has been monitored using either MRI and bioluminescence imaging in models of cerebral and cardiac ischemia or in brain tumor models. While phagocytotic cells such as monocytes can be efficiently labeled in situ, other cell types have to be harvested and labeled in vitro. Alternatively, genetically engineered cells expressing a reporter gene have been applied. While for most molecular imaging approaches the use of MRI is limited by its low intrinsic sensitivity, cells tolerated relatively high amounts of superparamagnetic iron oxide; therefore low amount of cells, in favorable cases even single cells, can be detected with unsurpassed spatial resolution.

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