Typical drug discovery processes utilize high-throughput methods to identify promising compounds followed by animal studies to examine efficacy and toxicity. This is a costly and high-risk venture, and the time spent on assessing compounds that ultimately fail toward the end of this process contributes to the rapidly escalating cost of bringing a drug to market.
An inherent problem in this approach is that it often identifies promising compounds without revealing mechanisms of drug action or mechanisms of cell toxicity early in this process. The application of high-content analytical (HCA) methods has partially addressed this problem, although current HCA methods are limited to cell-culture studies that cannot reproduce the complex cellular and systemic interactions that occur in vivo. Consequently, cell culture experiments often lack clinical and biological relevance.
Animal studies provide clinically relevant data that better predicts clinical outcomes. These studies generally suffer from difficulties in analyzing the cellular response to pharmaceutical agents. This shortcoming complicates the interpretation of the mechanisms underlying physiological and toxicological effects.
Bridging the gap between assessing mechanisms of efficacy and toxicity in animals is of vital importance in the drug discovery pathway. INphoton has developed a novel approach to close this gap, intravital multiphoton microscopy, which utilizes multiphoton fluorescence imaging (MFI) to provide a high-content, high-resolution analytical method for assessing drug efficacy and toxicity in animals.
MFI advances standard fluorescence imaging by utilizing short bursts of focused infrared light to provide spatially localized fluorescence excitation. The reduced toxicity and enhanced tissue penetration of infrared light allows MFI to operate as an in vivo imaging modality that can provide a combination of resolution and sensitivity at subsecond timescales.
Pharmaceutical companies are employing in vivo imaging to a greater extent as a means of evaluating the distribution and functional effects of drugs in living animals. Current in vivo imaging techniques such as magnetic resonance imaging, positron emission tomography, computed tomography, and whole-body fluorescence techniques, are capable of characterizing physiological processes (functional imaging) and the tissue distribution of drugs and their targets (molecular imaging).
The limited sensitivity and spatial resolution of these techniques, however, limits their ability to resolve critical events occurring at cellular and subcellular levels. Consequently, the current methods of in vivo imaging employed in drug development are incapable of resolving the cellular distributions and dynamic effects of drugs that are frequently critical to understanding their physiological actions and toxic side effects. In vivo MFI fills this void by providing a tool for collecting fluorescence images of living animals at a subcellular resolution.
As shown in Figure 1, in vivo MFI provides nanomolar sensitivity at submicron resolution. For the large number of drug compounds whose distribution in tissues cannot be preserved for histological evaluation, MFI provides a means of identifying the cells and subcellular compartments where experimental drugs accumulate. This data is frequently crucial to understanding drug efficacy and toxicity.
In addition, in vivo microscopy techniques capture images at 1–30 frames per second. The spatial and temporal resolution make in vivo MFI capable of characterizing the dynamics of drug transport at the cellular and subcellular level.
Figure 2 demonstrates the high-content, high-resolution capability of MFI as an in vivo imaging modality. It shows an in vivo multiphoton fluorescence image of the kidney from a healthy living rat collected minutes after intravenous injection of fluorescent-labeled compounds including gentamicin (red), a large molecular weight probe (green), and a probe to label the nucleus (blue). Important aspects of tubular and subcellular drug distribution are apparent in the glomerular filtration and subsequent tubular reabsorption of gentamicin via endocytosis by cells comprising the proximal tubule.
The resolution of this image shows not only that gentamicin uptake is limited to proximal tubule cells, but also, that gentamicin accumulates in endosomes and lysosomes of proximal tubule cells. By collecting images in time series, the kinetics of cellular accumulation of gentamicin can also be quantified (Figure 3).
Essential physiologic processes are also evident in Figure 2. These processes can be quantified and the alteration of these processes can serve as a marker of drug efficacy or toxicity. For instance, the unfiltered large molecular weight probe (green) is retained within the vasculature and effectively defines the vascular space (arrow). Microvascular perfusion rates can be determined by the geometry of red blood cell “shadows,” which form voids in the signal of the large probe coursing through the capillaries.
Integrity of the microvascular endothelial barrier is reflected by the complete retention of the large probe in the vasculature and the absence of leakage into the interstitial space. Furthermore, the absence of signal voids from white blood cells adherent to or rolling along the microvascular wall demonstrates the lack of leukocyte binding to the endothelium or leukocyte activation characteristic of an inflammatory process.
Finally, the nuclear label serves as a monitor for processes such as nuclear fragmentation that would herald apoptosis and cell death. As mentioned above, these quantifiable physiological readouts of organ function, vascular function, inflammation, and cell injury can be used to detect highly localized drug toxicities in a variety of organs that are otherwise undetectable in conventional assays. More importantly, the ability to spatially correlate physiological readouts with cellular and subcellular distributions of drugs gives investigators the ability to detect drug efficacy and/or toxicity at the level of individual cells. This level of resolution can provide drug developers with vital insights into drug actions.
Intravital multiphoton microscopy offers the advantages of both microscopic tissue analysis and in vivo imaging. As with microscopic analysis of histological tissue samples, intravital multiphoton microscopy provides subcellular resolution of multiple parameters. As with other forms of in vivo imaging, intravital multiphoton microscopy is free from fixation artifacts and supports dynamic studies and longitudinal studies of single animals over time. The synergy of these capabilities results in an investigational tool with capabilities that can complement and extend traditional ADMET approaches in drug development.
Timothy Alan Sutton, M.D., Ph.D., is CSO, and Kenneth William Dunn, Ph.D., is vp, scientific development, at INphoton. Web: www.inphoton.com. E-mail: firstname.lastname@example.org.