Monday, June 15, 2009

The evolution of live animal imaging technology has helped researchers get a better picture of the drug research process, from target identification to pharmacokinetic studies.


By Stephen Oldfield, PhD, Marketing Director, Imaging Products, Caliper Life Sciences

Prostate tumor visualized in 3D reconstructions for precise quantitation and localization. (Source: Caliper Life Sciences)

As animal imaging techniques evolve from providing information about anatomy to providing molecular-level detail about pathways and targeting, the utility of these applications has dramatically improved the drug development process for cancer research. Live animal imaging can be used in all stages of oncology drug development, from target identification through efficacy studies and disease phenotyping to the pharmacokinetic studies pre-Phase 1. Understanding a therapy's potential benefit from an early stage through detailed imaging applications can guide the evolution of a therapy and improve the likelihood of developing a successful drug candidate.

Oncology is one of the most prominent areas of scientific research, with more than $75 billion spent by the US government on cancer research over the past 30 years. Imaging applications are becoming increasingly important in oncology research as they are able to provide significantly more information at a lower cost to the researcher while using fewer animals. With a variety of imaging techniques available, it is most beneficial to utilize several modalities in a development process according to their relative strengths. Co-registering imaging data collected from multiple modalities can validate drug development and ultimately provide a more seamless path to the clinic.

Anatomical vs. Molecular Imaging

Live animal imaging methods are generally divided into anatomical and molecular techniques. The physical techniques like X-ray computed tomography (CT), ultrasound, or magnetic resonance imaging (MRI) are characterized by exquisite spatial resolution. Molecular imaging modalities such as positron emission tomography (PET), single photon emission computed tomography (SPECT), or optical methods may have lower spatial resolution but have evolved to characterize and quantify biological processes at the cellular and sub-cellular level. For drug development, the insight into functional mechanisms gained from molecular imaging is essential, but a combination of methods using multimode detection (e.g., PET-CT) or co-registration post-acquisition (e.g., optical-MRI) can provide additional insight and ultimately form the basis of translation to the clinic.

Physical imaging methods require structural changes large enough to be detected. With a molecular technique, however, an imaging probe can be used to interact directly with the molecular target, or a reporter gene linked to target expression can be monitored at very low levels of expression—or combinations of the above.

While target validation provides the first application area, the major economic benefits can be seen in efficacy studies where the ability to model multiple parameters at once enables more cycles of compound optimization in a given time frame. Further down the development pipeline, pharmacokinetic studies can also be accelerated using animal imaging techniques. With reporter gene assays performed in vivo, results can be generated in a few hours, avoiding an extensive histopathology analysis and weeks of delay for an actionable result.

Co-registration of CT and fluorescence images in 3D. (Source: Caliper Life Sciences)

Reproducible Results

Studies performed non-invasively in the intact animal allow for repetitive and reproducible analysis over a time course. Repeatedly imaging the same animals in a longitudinal study allows researchers to achieve statistical significance with relatively few animal subjects and generate a mechanistic understanding of the biological response. The savings in time and animal handling are significant, and the researcher avoids having to sacrifice animals at every time point. With optical reporters, the method is not only sensitive, but can also be high-throughput and low cost. For oncology research in particular, transgenic tumor lines can be readily engineered with fluorescent or bioluminescent reporters to provide a high throughput platform and a low cost assay.

Fluorescence and Bioluminescence

Fluorescent and bioluminescent optical imaging have developed as sensitive, cost-effective methods for quantitative, noninvasive longitudinal studies monitoring tumorigenesis, metastasis, and responsiveness to anticancer agents in living animals. Fluorescence allows the visualization of both metabolically active and inactive cells in vivo, and also provides a convenient histological marker. Light emission requires excitation by an external light source. The typical approach is to genetically encode luciferase or fluorescent proteins (red fluorescent proteins work best) into tumor cells so that the molecular or cellular events can be visualized and tracked non-invasively during cancer disease progression.

Bioluminescence typically monitors metabolically active cells and requires interaction of the luciferase with injected luciferin and ATP. It can be exquisitely sensitive in the detection of labeled cells and can visualize a single cell subcutaneously. Both fluorescent and bioluminescent moieties can also be conjugated to endogenously injected targeting probes in order to detect cellular markers in vivo.

Orthotopic Oncology Models

Non-invasive, in vivo optical imaging using bioluminescent reporters enables an entire field of orthotopic oncology models. Compared to subcutaneous models of tumor development, orthotopic models can show more relevant host-tumor interaction with organ-specific growth environment and drug exposure, characteristic disease progression, metastatic potential, and response to therapy. Those differences reflect the greater biological relevance of the model. Orthotopic tumors are more likely to metastasize earlier, and metastatic progression contributes significantly to the survival time. Live animal imaging provides oncology researchers with means to explore orthotopic and spontaneous tumors that more closely represent natural tumor development.

Accelerating the Drug Discovery Process

Prostate tumor imaged using bioluminescence. (Source: Caliper Life Sciences)

With an appropriate biological model and an efficient imaging method, live animal studies can be used to screen compounds early in the development process, enabling additional cycles of optimization on successful drug candidates to reduce their risk of failure at a later, more costly stage. Getting compound into animals sooner allows researchers to more quickly identify risks. In addition, the increased speed of assays allows researchers to run additional chemistry and efficacy models before progressing to the next phase. A low-cost assay method in live animals, therefore, significantly accelerates the discovery process and improves the quality of the lead compounds being carried forward.

Monitoring Tumor Growth

Cancer metastases are the most lethal attributes of human malignancy and remain an ongoing therapeutic challenge for drug discovery. Using bioluminescent imaging, tumor development can be monitored, non-invasively, directly after implantation and micrometastases can be monitored from a minimal number of cells and followed throughout development concurrently with primary tumor growth.

From a practical perspective, bioluminescent imaging (BLI) can be used to randomize animals into experimental cohorts before establishing the treatment regimes. Animals can be monitored routinely from day one to follow development of the primary tumors—whatever their location—without the need for sacrifice, dissection or histopathology. By following the same animal longitudinally, the kinetics of metastatic burden can be easily monitored throughout treatment.

Drug treatment, regression and relapse can also be measured in the same cohort of animals, generating improved statistics from fewer animals. With BLI it is easy to measure the anti-tumor and anti-metastatic activity of a therapeutic intervention—even if the activity can not be detected by physical means.

Pathways and Mechanisms

Because BLI output depends on the presence of actively metabolizing cells it can be more sensitive in the detection of therapeutic impact than physical measurement of a tumor. Drug therapies that kill tumor cells will cause an immediate reduction in BLI, but if they do not cause shrinkage of the tumor they can be missed in screening. Optical imaging techniques also allow the exploration of underlying mechanisms of tumor growth, with models for the monitoring of hypoxia, apoptosis, angiogenesis, or gene regulation in vivo and in real time.

Bioluminescent reporters are commonly used to monitor VEGFR2 expression when angiogenesis is stimulated during tumor growth. The tumor itself may be expressing a fluorescent protein and fluorescent conjugates are commonly used to track specific tumor markers such as HER2 on the cell surface. So called "Active" dyes change their fluorescent emission when they are chemically modified in situ (e.g., cleaved by proteases), and with careful selection of their spectral properties, all of these optical signals can be imaged together to unravel the complex biological processes.

Three-Dimensional Data

Planar, two-dimensional imaging is an ideal tool early in drug discovery because it is low cost, high throughput, rapid to perform and quantitative for longitudinal studies. For additional insight into the disease process, or for absolute quantitation of a signal, an optical tomographic method can be used to generate three-dimensional data output. Both bioluminescence and fluorescence can be imaged in three dimensions and the reconstructed images can be co-registered with other imaging modalities. Reconstruction of BLI with CT, for example, might be critical in demonstrating a tumor metastasizing to bone. Co-registering fluorescence with MRI might be integral to developing a translational contrast agent for use in the clinic.

Conclusion

Access to live animal imaging has provided key results for judging the efficacy of many drugs in the clinic or in clinical trials. Live molecular imaging provides data consistent with results from physical measurements, and histology or anatomical imaging techniques such as X-ray or MRI while providing the least amount of disruption to normal biological processes. The robust, quantitative and high-throughput modality of optical imaging provides an ideal platform for pre-clinical development. Using optical imaging techniques, tumor development can be followed with a rigor that would be unfeasible using traditional sampling and histology techniques and can be more stringently evaluated for compound efficacy, providing additional validation and a direct path to the clinic.
n Stephen Oldfield received his PhD from Imperial College, London and has worked in various tool companies providing innovative technology for drug discovery and development.

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