Noninvasive assessment of the target inhibition is important at various stages of drug development. In phase I studies, assessment of target inhibition can define the biologically active dose of a new drug for further phase II and III studies. Traditionally, the dose of a cancer drug chosen for further investigation has been determined by the maximum tolerated dose (MTD) in phase I studies. Pathway-targeted drugs, however, frequently cause limited acute adverse effects; the biologically active dose might be substantially lower than the MTD. Furthermore, classic cytotoxic agents are administered over a relatively brief period of time, with the intent to eradicate tumor cells. By contrast, many pathway-targeted drugs are developed as oral treatments for long-term use and are designed to inhibit tumor growth. Dosing these drugs at the MTD might result in unnecessary toxicity. Moreover, the effectiveness of some targeted drugs may actually decrease at higher doses, owing to off-target effects on other biologically active molecules.
In phase II studies, new drugs commonly fail to produce the desired responses for two reasons. First, the dose schedule might be inadequate; in other words, the target might not be inhibited. This fact should not, however, lead to ‘no go’ decisions; instead, the dose or schedule of administration must be optimized. Second, drugs might inhibit the target but might not affect tumor growth if the target is not essential. This should lead to ‘no go’ decisions.
If the probes used in imaging bind to the same target as the drug (), inhibition of the target can be monitored by imaging the blocking of probe uptake by the study drug.33,34
Similarly, inhibition of enzymatic activity can also be visualized by imaging probes that are substrates of specific enzymes. This approach has been used to monitor the inhibition of matrix metalloproteinase activity by activatable MRI probes that are cleaved by this enzyme.35
Figure 3 Monitoring of target inhibition by PET imaging. (A) Blockade of αVβ3 integrin by the cyclic pentapeptide c(RGDfV). Mice were implanted with an αVβ3-positive tumor on the left shoulder. Pretreatment with the αVβ (more ...)
Few ligands have been validated for imaging target inhibition in this way. Also, radiolabeling of drugs frequently does not provide useful imaging probes for monitoring target inhibition because uptake of the drug in the tumor can be dominated by nonspecific binding to the cell membrane or other cellular components. In addition, although the drug concentration does not need to be higher at the target relative to the surrounding tissues, this is a necessary condition for imaging probes. Finally, whether the kinetics of the drug are favorable for imaging and whether analysis of the image is confounded by radiolabeled metabolites must be determined.36
If a probe is unavailable for a known target, imaging of biochemical or biological changes in response to target inhibition can provide a surrogate for imaging the interaction between the drug and the target. Using this approach, Smith-Jones et al.
studied levels of expression of HER2 in murine xenograft models of breast cancer in response to treatment with the heat shock protein 90 (Hsp90) inhibitor 17-allylaminogeldanamycin (17-AAG).37
HER2 expression was measured by use of micro-PET with [68
Ga]trastuzumab and a [68
Ga]trastuzumab F(ab') 2 fragment (because of more rapid blood clearance of this fragment than of trastuzumab). Both imaging probes showed high retention in tumors, which correlated to HER2 expression by immunoblotting. Serial micro-PET imaging to measure the effects of 17-AAG on the levels of HER2 expression in tumors is feasible because 68
Ga has a short half-life of only 68 min and the F(ab') fragment is cleared quickly from the circulation. Treatment resulted in a reduction of more than 50% in probe uptake in the tumor 24 h after 17-AAG treatment (). Expression of HER2 measured in excised tumor tissue was reduced by 80% compared with that in vehicle-treated controls. The binding of the probe to the target was specific because 17-AAG treatment did not affect the levels of phosphatidylinositol 3-kinase, an Hsp90-independent protein. The same group demonstrated that the 17-AAG-dependent degradation of HER2 did not result in reduced tumor size or tumorglycolysis, by use of micro-PET analysis of 2-deoxy-2-[18
F]fluorodeoxy-D-glucose (FDG) uptake.38
These studies have several potential implications for human trials. First, the [68Ga]trastuzumab F(ab') 2 fragment (or other appropriately engineered antibody fragments) could be used to identify breast cancer patients who overexpress HER2 and are, therefore, more likely to respond to treatment with trastuzumab. Second, an early reduction in [68Ga]trastuzumab F(ab') 2 fragment binding could distinguish effective 17-AAG treatments in vivo. Third, PET imaging might elucidate differences in HER2 expression in distinct metastatic lesions. Fourth, PET imaging might be used to determine noninvasively whether the MTD of the drug can inhibit the therapeutic target. Finally, the dose scheduling could be optimized by monitoring the duration of target inhibition using PET.
Dynamic contrast-enhanced (DCE-) MRI has also been used to study the effects of inhibitors of VEGF signaling. VEGF markedly increases vascular permeability; monitoring changes in vascular permeability presents an attractive approach to the imaging of target inhibition by anti-VEGF antibodies or VEGF receptor kinase inhibitors.39
DCE-MRI was used to determine the effective dose for the pan-VEGF receptor kinase inhibitor PTK 787/ZK 222584 (PTK/ZK). Treatment with this drug resulted in a dose-dependent decrease of Ktrans
, a marker of vascular permeability and perfusion. The effects of the drug on Ktrans
plateau at a dose of 1,000 mg/day.40
The MTD of PTK/ZK is 1,500 mg/day and, therefore, the biologically effective dose seems to be significantly lower than the MTD; DCE-MRI was instrumental in determining the appropriate dose ().41
Figure 4 Monitoring of target inhibition by dynamic contrast-enhanced MRI. At baseline (top row), the multiple liver metastases (arrows) demonstrate intense contrast enhancement. Following treatment with the VEGF receptor protein kinase inhibitor PTK/ZK, the liver (more ...)
Molecular imaging can be particularly helpful in dose-finding studies of anti-angiogenic agents because the genetic variability of the endothelial cells forming the blood supply to the tumor is likely to be smaller than that of the tumor cells. As a consequence, a relatively low interpatient variability of the drug concentration needed for endothelial cell target inhibition would be expected. By contrast, genetic variation of tumor cells can cause marked differences in their sensitivity to targeted drugs. For example, mutated forms of the EGFR found in some NSCLCs can be inhibited by EGFR kinase inhibitors at concentrations approximately 100-fold lower than those required to inhibit wild-type EGFRs.42
Such marked genetic variations in drug sensitivity can complicate the establishment of dose–response relationships in phase I studies.
Although DCE-MRI has shown encouraging data for imaging of target inhibition by PTK/ZK and other VEGF inhibitors, several technological challenges remain. Acquisition and analysis of DCE-MRI scans are technically demanding and not uniformly standardized. In clinical studies, the quality of data is frequently inadequate for quantitative analysis.43
There is also considerable intrapatient variability of the quantitative parameters; two pre-therapy baseline scans are currently recommended to assess the reproducibility of measurements in clinical trials.43
It is also unclear whether DCE-MRI will be as successful as existing imaging techniques for the assessment of the effects of drugs that are less potent inhibitors of vascular permeability than are VEGF inhibitors.
In the experimental setting, molecular imaging with genetically encoded reporters can provide additional insights into ligand–target interactions. In the ‘split protein’ strategy,44
a reporter enzyme is cleaved into N-terminal (Nr
) and C-terminal (Cr
) fragments, each of which is enzymatically inactive. Each fragment is fused to one of two interacting proteins (X and Y). A physical interaction between Nr
-X and Cr
-Y proteins reconstitutes the activity of the enzyme, leading to the generation of a signal after substrate administration. Thus, interactions between X and Y proteins mediated by drug administration can be studied in vivo
. This approach has been used to study protein–protein interactions induced by the mTOR inhibitor rapamycin, which mediates the interaction of the FRB and FKBP12 proteins. The resulting FRB–rapamycin–FKBP12 complex inhibits mTOR, a serine–threonine kinase. To monitor this process in vivo
, fragments of firefly luciferase were fused to FRB and FKBP12. Rapamycin-induced formation of the FRB and FKB12A complex restores the enzymatic function of luciferase; following injection of luciferin, light emission can be monitored noninvasively by optical imaging.45,46
Although studies involving genetically encoded reporters cannot be used in the clinical setting, they can provide fundamental information on the ability of new drugs to target protein–protein interactions in animal models. These reporters might be used for high-throughput screening of drugs in cell culture assays.