Biomedical Engineering Reference
In-Depth Information
a radionuclide, a l uorescent molecule, or an MRI contrast agent. It is important that the labeling
procedure preserved the pharmacophore of the receptor ligand, i.e., the part of the molecule that
is critical for its interaction with the target. The development of such specii c reporter constructs
has to tackle the same issues encountered in the development of a therapeutic: (1) the probe has to
be target-specii c with minimal cross-reactivity to other receptor systems; (2) it must have good
pharmacokinetic (PK) properties, i.e., the probe should be able to penetrate tissue barriers to reach
the target and the exposure time has to be long enough to allow for the target-specii c interaction,
and the nonreacting fraction of the label should be cleared rapidly from the system to maximize
signal-to-background ratio (SBR); (3) the reporter construct should be biocompatible, not posing
any safety issues; and (4) in addition as the concentration of targets in tissue is generally low, the
signal generated by the reporter group should be amplii ed when possible. This can be achieved
by increasing the payload, by ligand trapping, or enzymatic probe processing. So-called target-
activatable probes constitute an elegant strategy to improve SBR. These are reporter moieties that
change their biophysical properties upon interaction with their target. Fluorescent molecules or
MRI contrast agent qualify for such designs, as these l uorescence and magnetic dipole interac-
tions can be modulated by the local environment. Enzyme activatable probes fall into this category.
A critical issue with exogenous probes is their delivery to the target, in particular when the target
is located intracellular. Cellular uptake of low molecular weight probes is optimized by derivatiza-
tion following strategies such as Lipinski's rule of i ves. For larger probes, target delivery may be
ach ieved by exploiting cellula r upta ke mecha n isms such as tra nspor ter systems or receptor-mediated
endocytosis or by conjugation of cell-penetrating peptides to the reporter moiety.
Reporter gene assays
: Complementary to the use of exogenous probes, reporter assays generated by
the biological system itself might be used. Reporter gene assays are established tools in molecular
and cell biology. They involve the generation of a genetically modii ed cell (or animal) that expresses
a reporter molecule under the control of the promoter of the gene of interest, or use a strategy, in
which the target protein drives the expression a reporter gene. A remarkable number of reporter gene
assays suited for application in whole animals meanwhile suitable for MRI, PET, l uorescence imag-
ing have been developed the best known being l uorescent proteins such as green l uorescent protein
(GFP) or bioluminescent proteins such as luciferases. It is obvious that these systems are largely lim-
ited to animal studies (except for gene therapy approaches or potentially cell tracking in humans).
7.2.2.2 Molecular Imaging Strategies, Imaging Targets
Imaging approaches may target different aspects that relate to the efi cacy of drugs: its biodistribu-
tion, 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 biodistri-
bution is of key importance in the development of novel therapeutics. During the preclinical
phase this information is commonly obtained from quantitative whole-body autoradio-
graphic 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 identii cation is
based on the mass determination; hence, MALDI-MS imaging does not require labeling
with radioisotopes. In addition, using the mass i lter 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
