This PDF file contains the front matter associated with SPIE Proceedings Volume 11219, including the Title Page, Copyright information, Table of Contents, Author and Conference Committee lists.

Regulatory agencies require that drug developers characterize the absorption, pharmacokinetics, distribution, metabolism, and elimination (ADME) characteristics of new small and large molecule therapeutic entities. Quantitative whole-body autoradiography (QWBA) has become the definitive tool used for the determining tissue distribution patterns and is specifically requested by the FDA for IND submissions. However, the use of imaging mass spectrometry (IMS) to examine tissue distribution of xenobiotics has quickly gained the attention of pharmaceutical researchers because it does not require the use of radiolabeled test compounds and it can specifically identify the test article and their known metabolites with high imaging resolution. This talk will describe the state-of-the-art of both techniques and present examples of their applications in drug discovery and development.

We introduce and validate a framework for imaging and quantifying active molecule penetration into human skin in vivo. Our approach combines nonlinear imaging microscopy modalities, such as two-photon excited auto-fluorescence (TPEF) and coherent anti-Stokes Raman scattering (CARS), together with the use of deuterated active molecules. The imaging framework is exemplified on topically applied glycerol diluted in various vehicles such as water and xanthan gel. In vivo glycerol quantitative percutaneous penetration over time is demonstrated, showing that, contrary to water, xanthan gel vehicle acts as a film reservoir that releases glycerol continuously over time.

There remains a paucity of methodological tools to determine the biodistribution of vaccine antigens. In response to this, we established a near-infrared (NIR) imaging method using a NIR fluorophore, ZW800-1C, conjugated with different sizes of vaccine antigens that allows for real-time monitoring of the fate of delivered vaccines in vivo. The fluorescent signal observed using the system after a model vaccine injection in mice recapitulated the size-dependent transport of the vaccine into the secondary lymphoid tissue. This methodology can be broadly applied for optimization of formulations and safety evaluation of clinical vaccines.

Scanning confocal Raman spectroscopy was applied for detecting and identifying topically applied ocular pharmaceuticals on rabbit corneal tissue. Raman spectra for Cyclosporin A, Difluprednate, and Dorzolamide were acquired together with Raman spectra from rabbit corneas with an unknown amount of applied drug. Kernel principle component analysis (KPCA) was then used to explore a transform that can describe the acquired set of Raman spectra. Using this transform, we observe some spectral similarity between cornea spectra and Cyclosporin A, with little similarity to Dorzolamide and Difluprednate. Further investigation is needed to identify why these differences occur.

Optical tomography is often coupled with high resolution imaging modality like MRI to provide functional information associated with specific anatomical structure noninvasively. MRI-coupled paired agent fluorescence molecular tomography (MRI-PAFT) is a hybrid imaging modality capable of noninvasively quantifying drug-target engagement in vivo utilizing a targeted and an untargeted fluorescence agent. This study compares the uptake kinetics of MRI contrast agent and fluorescence agents in tumor and normal tissue, and demonstrates the potential of utilizing MRI contrast agent kinetic and targeted fluorescence agent kinetics to quantify targeted tumor receptor concentration in glioma tumor model.

Human EGF receptor 2 (HER2) is an important oncogene and marker of aggressive metastatic cancer, found in up to 20% of oncologic patients. Anti-HER2 humanized monoclonal antibody trastuzumab (TZM) has been successfully used over the last two decades. However, both primary and acquired TZM resistance calls for the deeper investigation on TZMHER2 binding, internalization and trafficking/degradation in cancer cells in vitro and in vivo. Fluorescence lifetime FRET imaging (FLIM FRET) offers a unique approach to monitor TZM-HER2 binding followed by their uptake into target cells via the reduction of donor fluorophore lifetime. In this study, we characterized for the first time TZM-AF700 uptake and its relation to HER2 expression in AU565 human breast cancer cell line using confocal microscopy. Further, we have quantified the dimerization of HER2 via NIR FLIM FRET in vitro microscopy. Extensive analysis confirmed high specificity and efficiency of TZM FRET signal. Interestingly, we observed a significant heterogeneity of FRET within the cells: the highest TZM FRET levels occurred at the plasma membrane, whereas less if any donor lifetime reduction was registered in the perinuclear endosomes. These results suggest that HER2 dimers undergo dissociation or degradation upon TZM binding and trafficking. Overall, this study provides a good foundation for in vivo TZM FRET imaging of target engagement in preclinical studies.

Raman microscopy is a powerful tool to observe molecular distribution in live cells. Here, we propose a technique to detect small molecule drugs using alkyne-tag surface-enhanced Raman scattering (SERS) imaging. To obtain SERS effect, we use gold nanoparticles as SERS probes so that we can enhance Raman scattering of small molecule drugs at low concentration with several orders of magnitude. We use alkyne tag to selectively detect the drug molecules by using the Raman peak of alkyne in the spectral silent region. Home-built slit-scanning Raman microscopy enables us to perform rapid SERS imaging. We successfully detect SERS signal from an alkyne-tagged inhibitor of a lysosomal enzyme with gold nanoparticle modified glass substrate.

Intracellular Paired-Agent Imaging (iPAI) quantifies intracellular drug targets using fluorescently-labeled small molecule imaging agents. iPAI has the potential to predict therapeutic response for individual patients but requires a patient derived xenograft (PDX) model that accurately mimics in vivo tumor heterogeneity yet is easily accessible for intravenous drug administration and rapid image collection. The chicken embryo chorioallantoic membrane (CAM) provides an intermediate, cost-effective model that provides vascularized, in vivo tumors in a matter of days (typically ~72 h) from implantation. Here, we investigate the implantation of thin (< 1 mm) cross-sectional slices (1-2 cm) of freshly excised tumor tissues from mouse xenografts. Multispectral iPAI will be performed to quantify the distribution and heterogeneity of drug targets within the tumor cross-section.

Targeting the aberrant epidermal growth factor receptor (EGFR) signaling pathway is an attractive choice for many cancers (e.g., non-small cell lung carcinoma (NSCLC) and head and neck squamous cell carcinoma (HNSCC)). Despite the development of promising therapeutics, incomplete target engagement and acquired resistance (e.g., mutagenesis and intracellular signaling pathway rewiring) ensure that curative options still elude patients. To address limitations posed by standard drug evaluation assays (e.g., western blot, bulk plasma monitoring, immunohistochemistry), we have developed a novel dynamic, fluorescence-based platform termed intracellular paired agent imaging (iPAI). iPAI quantifies intracellular protein target engagement using two matched small-molecule, cell membrane-permeable agents: one targeted to the protein of interest and one untargeted, which accounts for non-specific therapeutic uptake. Currently, our iPAI panel includes successfully characterized tyrosine kinase inhibitors targeting the kinase binding domain of numerous proteins in the EGFR pathway, including erlotinib (EGFR). Here, we present a pharmacokinetic uptake study using our novel iPAI erlotinib reagents: a targeted erlotinib probed conjugated to silicon tetramethylrhodamine (Erl- SiTMR-T) and an untargeted reagent conjugated to tetramethylrhodaime (Erl-TMR-UT). An initial uptake study in a cell derived xenograft (CDX) model of NSCLC was performed by administering the Erl iPAI reagents systemically via tail vein injection, where drug uptake was quantified in the tumor over time. Excitingly, evidence of heterogeneous uptake was observed in the iPAI injected cohort, displaying distinct drug-uptake within a single tumor. Characterization of additional iPAI agents targeting downstream effectors (e.g., AKT, PI3K, MEK and ERK) is ongoing and will allow us to visualize complex drug-target interactions and quantify their downstream signaling partners during treatment regimens for NSCLC and other cancers. Together, we anticipate these iPAI probes will improve understanding of current limitations in personalized cancer therapy.

A paired-agent fluorescent molecular imaging strategy is presented as a method to measure drug target engagement in whole tumor imaging. The protocol involves dynamic imaging of a pair of targeted and control imaging agents prior to and following antibody therapy. Simulations demonstrated that antibody “drug target engagement” can be estimated within a 15%-error over a wide range of tumor physiology (blood flow, vascular permeability, target density) and antibody characteristics (affinity, binding rates). Experimental results demonstrated the first in vivo detection of binding site barrier, highlighting the potential for this methodology to provide novel insights in drug distribution/binding imaging.

MRI images of gadolinium-based contrast agents (GBCA’s) acquired before surgery are often registered to patients and used to guide surgical resection of intracranial tumors. Yet, the accuracy of these MR images in describing the surgical field degrades as surgery progresses; a well-recognized problem which has prompted efforts to develop new techniques that provide updated guidance information on residual tumor location. These efforts span a wide array of technologies, including image updating with deformation models, intraoperative MRI, and fluorescence guided surgery, among others. However, introduction of a straightforward technique that provides surgeons with a current view of GBCA distribution in real time remains an important goal. In this context, development of a fluorescent agent that recapitulates the kinetic behavior of GBCA’s could provide familiar information directly in the surgical field in real time. To advance this strategy, we have begun identifying fluorescent contrast agents that show similar kinetic behavior to GBCA’s. Using a novel hyperspectral whole body cryo-imaging system, we acquired highresolution 3-D volumes of the distribution of multiple candidate fluorophores in whole heads bearing orthotopic brain tumors. Preliminary results reveal significant differences in the distribution of candidate optical agents, some of which show strong similarity to the GBCA uptake. Identification and eventual translation of a reliable GBCAoptical analog could improve and simplify surgical resection of brain tumors.

Glioblastoma has a high rate of recurrence due to treatment methods often failing to penetrate the blood brain barrier. To overcome this limitation, photodynamic priming (PDP) can be used to increase tissue permeability. In this study we investigate the feasibility of using fluorescence laminar optical tomography (FLOT) to provide quantitative distribution information on photodynamic drug in the brain to optimize the timing of PDP. The project will result in a non-invasive way to quantify the concentration of photodynamic drug in the brain. This would allow for optimized treatment times, leading to improved patient outcomes.

Small molecule kinase inhibitors (SMKIs) drugs have the potential to offer exquisite specificity in controlling aberrant intracellular signaling pathways in cancer and other disease states. However, while nearly 50 SMKIs have been FDA-approved, patient responses have been variable, and sensitive populations not easy to identify. For instance, in non-small-cell lung cancer, only 30% of patients respond to the epidermal growth factor receptor (EGFR) targeted SMKI, erlotinib, yet the level of erlotinib uptake is a poor indicator of treatment efficacy. The development of fluorescently-labeled SMKIs that maintain their viability as drugs has facilitated the use of paired-agent molecular imaging protocols that are able to discriminate, in vivo, between imaging agent uptake and binding. Here we present a mathematical framework of SMKI transport and binding, in vivo, and derive a kinetic model for extracting SMKI binding potential (BP) from kinetic fluorescent-SMKI imaging data-proposed as a more effective indicator of potential therapeutic response than SMKI uptake alone. The accuracy and precision of the SMKI BP kinetic model was demonstrated in simulation studies and in an in ovo xenograft experiment. In simulation, the SMKI BP estimates were within 20 􀀀 5% of expected values over a large range of physiologically relevant blood flow, vascular permeability and cell permeability; and over a range of SMKI affinity, cell membrane permeability, and blood plasma pharmacokinetics. The in ovo experiment bolstered the simulation findings, demonstrating a statistically significant spatial correlation (r > 0.9, p < 0.01) between EGFR concentration measured by a validated extracellular approach and the SMKI BP approach.

Chemotherapeutics are generally delivered systemically, causing damage to rapidly dividing cells and leading to a compromised immune system, nausea, fatigue, and hair loss. In situ forming implants (ISFIs) are biocompatible drug delivery vehicles which are injected as a liquid, undergo phase inversion during drug delivery, and ultimately breakdown, thereby allowing for localized release of drugs, potentially reducing side effects of chemotherapeutics.

Previous experiments in our lab have shown that applying one-time ultrasound to ISFIs loaded with chemotherapeutics, shortly after injection, the 8-day release of doxorubicin can increase from 60% to 95% in vivo. To separate the effects of sonication on the effective drug diffusion rate from those related to how the US exposure may change the phase inversion kinetics and drug release, ISFIs and reservoirs of Janus Green B dye, located approximately 6 mm apart, were embedded in a polyacrylamide phantom. ISFIs were stimulated with a 1 MHz focused transducer with either no, mild (10 min, 10% duty), or intense (5 min, 33% duty) sonication, and phantoms were imaged to determine diffusion rates.

The relative diffusion area and dye diffusion rate were analysed as a function of time, regions enclosed by a thresholding contour line and sonication type. Compared to the no sonication control, the effects of mild sonication were variable, with relative area increasing in low concentration contours and decreasing at high concentration contours (p = 0.0002). Intense sonication, compared to no sonication, had increased dye diffusion rate (p= 0.014), and a decrease in the relative area at early time points, and an increase at later time points (p = 0.041).

The results suggest that high intensity ultrasound exposure on a nearby object can cause changes to the diffusion of molecules located in the proximity of the object.

The ability to directly measure whole-body fluorescence can enable tracking of labeled cells, metastatic spread, and drug bio-distribution. We describe the development of a new hyperspectral imaging whole body cryomacrotome designed to acquire 3-D fluorescence volumes in large specimens (whole animals) at high resolution. The use of hyperspectral acquisition provides full spectra at every voxel, enabling spectral decoupling of multiple fluorohpores and autofluorescence. We present examples of tissue spectra and spectral fitting in a rodent glioma xenograft.

Successful cancer treatment continues to elude modern medicine and its arsenal of therapeutic strategies. Therapy resistance is driven by significant tumor heterogeneity, complex interactions between malignant, microenvironmental and immune cells and cross talk between signaling pathways. Advances in molecular characterization technologies such as next generation sequencing have helped unravel this network of interactions and identify druggable therapeutic targets. Tyrosine kinase inhibitors (TKI) are a class of drugs seeking to inhibit signaling pathways critical to sustaining proliferative signaling, resisting cell death, and the other hallmarks of cancer. While tumors may initially respond to TKI therapy, disease progression is near universal due to mechanisms of acquired resistance largely involving cellular signaling pathway reprogramming. With the ultimate goal of improved TKI therapeutic efficacy our group has developed intracellular paired agent imaging (iPAI) to quantify drug target interactions and oligonucleotide conjugated antibody (Ab-oligo) cyclic immunofluorescence (cycIF) imaging to characterize perturbed signaling pathways in response to therapy. iPAI uses spectrally distinct, fluorescently labeled targeted and untargeted drug derivatives, correcting for non-specific drug distribution and facilitating quantitative assessment of the drug binding before and after therapy. Ab-oligo cycIF exploits in situ hybridization of complementary oligonucleotides for biomarker labeling while oligonucleotide modifications facilitate signal removal for sequential rounds of fluorescent tagging and imaging. Aboligo CycIF is capable of generating extreme multi-parametric images for quantifying total and phosphorylated protein expression to quantify protein activation, expression, and spatial distribution. Together iPAI and Ab-oligo cycIF can be applied to interrogate drug uptake and target binding as well as changes to heterogenous cell populations within tumors that drive variable therapeutic responses in patients.

Long-term survival of head and neck squamous cell carcinoma (HNSCC) patients have proven to be correlated with negative surgical margins. Paired-Agent Imaging for Resection during Surgery (PAIRS) is capable of drawing the fine line between tumor and normal tissue by employing a control imaging-agent, which is co-administered with the targeted imaging agent to account for nonspecific signal. PAI is highly dependent on the parallel paired-agent delivery and static quantum yield of the agent to trace the molecular concentration. However, it is well known that nonspecific binding of fluorescence probes to plasma proteins can change its delivery, dissociation constant, and quantum yield.

A thorough evaluation of the effect of plasma protein binding in the estimation of receptor concentration was performed for the paired-agents in this study. We are planning to evaluate ABY-029, an anti-epithelial growth factor receptor (EGFR) Affibody, and IRDye 700DX as a control agent. The plasma-dependent change in fluorescence intensity, percent binding, and in vivo distribution kinetics will be studied for each agent alone, and in combination. In this proceeding, the absorption, emission patterns for the targeted agent, ABY-029, measured by UV-Vis, fluorometer, and Pearl were shown. Initial studies indicate that binding to Bovine serum albumin (BSA), human serum albumin (HSA) and EGFR can introduce the Solvatochromic shift, which will change the absorption and emission pattern for ABY-029. Computational modeling will be performed to determine how each of these changes will affect the determined BP, and thus detection of tumors from normal tissue.