This PDF file contains the front matter associated with SPIE Proceedings Volume 10859, including the Title Page, Copyright information, Table of Contents, Author and Conference Committee lists.

The ability of a drug molecule to reach the right protein in the correct intracellular compartment of the target cell type, in the desired tissue following a systemic dose, is governed by a complex network of active and passive transport processes. Furthermore, the same biological mechanisms that ensure on-target exposure are also responsible for the delivery of drug to off-target sites, potentially causing negative outcomes. Understanding the concentration of drug at target (D@T) is therefore one of the “3 pillars of successful drug discovery” [Morgan et al 2012]. The understanding of D@T can be further characterised by three components; the precise location (geography), the accurate concentration (Maths) and the chemical identity of the molecule (Chemistry) at scales ranging from whole body in the clinic to ex vivo tissue samples to single cells in culture. To that end GSK have harnessed a wide spectrum of analytical techniques utilising radioactive tracers, mass spectroscopy detection and optical methodologies, often in partnership with external academic and governmental collaborators.. This presentation will review the challenges of determining D@T in the diverse GSK portfolio and highlight the opportunities that Biophotonic methods, such as Raman and CARS, bring to our work. Finally we will review the key contribution of the external academic network to Bioimaging at GSK and share a few of our future challenges.

The determination of the absorption, pharmacokinetics, distribution, metabolism, and elimination (ADME) characteristics of new small and large molecule therapeutic entities plays a major role in the drug discovery and development processes. Combinations of new and old detection systems in drug research has advanced the discovery and development small molecule drugs and biotherapeutics. The data obtained from microautoradiography (MARG), quantitative whole-body autoradiography (QWBA), cryo-imaging, and imaging mass spectrometry (IMS) can be coupled with in vivo imaging data to offer high resolution answers to numerous questions regarding ADME properties of therapeutic compounds, such as: organ-tissue-cellular localization, receptor-specific localization, subcellular localization, drug interactions, gene expression, formulation comparisons, nanoparticle tracking, stem cell migration, virus localization, tissue metabolite ID, pharmacokinetics, pharmacodynamics, and target organ/tumor penetration. These techniques have been used to quantitatively assess the ADME characteristics of small molecule drugs, radiopharmaceuticals, proteins, peptides, oligonucleotides, antibodies, anti-body drug conjugates, and siRNA. A brief description of ADME study designs, the state-of-the-arts, and examples of their applications will be presented here.

Low response rates in solid tumors including head and neck cancers (HNCs) have been attributed to failure of the drug to reach its intended target. However, investigation of drug delivery has been limited due to difficulties in measuring concentrations in the tumor and the ability to localizing drugs in human tissues. Factors determining intratumoral antibody distribution in primary tumor and metastatic lymph nodes have not been well-studied in human patients. To address this challenge, we propose to leverage fluorescently labeled antibodies to investigate antibody delivery into HNCs. To this end, we have conducted a first-in-human clinical trial to assess the delivery of panitumumab-IRDye800 in HNCs. Twenty-two patients enrolled in this study received intravenous administration of panitumumab-IRDye800 at multiple subtherapeutic doses: (1) 0.06mg/kg, (2) 0.5 mg/kg, (3) 1 mg/kg, (4) 50 mg flat dose, (5) 25 mg flat dose. To quantify the antibody delivery, fresh tumor samples were procured and the amount of antibody in the tumor was quantified as ng/mg of tissue, which was then correlated with tumor characteristics. Immunohistochemistry of multiple protein markers, including EGFR, ERG, cytokeratin, Ki67, alpha-smooth muscle actin, etc., have been implemented in serial sections of primary tumors and metastatic lymph nodes. A quantitative image analysis pipeline was developed to analyze these IHC images and score the staining on both global and local scale. A predictive model was built to identify the most important predictors for antibody penetration from pharmacological factors, tumor pathophysiological factors, and tumor microenvironmental factors.

There is an increasing need in cosmetic research for non-invasive, high content, skin imaging techniques offering the possibility to avoid performing invasive biopsies and to supply a maximum of information on skin state throughout a study, especially before, during and after product application. Multiphoton microscopy is one of these techniques compatible with in vitro and in vivo investigations of human skin, allowing its three-dimensional (3D) structure to be characterized with sub-μm resolution. Various intra-/extra-cellular constituents present specific endogenous two-photon excited fluorescence and second harmonic generation signals enabling a non-invasive visualization of the 3D structure of epidermal and superficial dermal layers. In association with fluorescence lifetime imaging (FLIM) and specific 3D image processing, one can extract several quantitative parameters characterizing skin constituents in terms of morphology, density and function. Multiphoton FLIM applications in cosmetic research range from knowledge to efficacy evaluation studies. Knowledge studies aim at acquiring a better understanding of appearing skin differences, for example, with aging, solar exposure or between the different skin phototypes. Evaluation studies deal with efficacy assessment of cosmetic ingredients in anti-aging or whitening domains. When using other nonlinear optics phenomena such as CARS (Coherent Anti-Stokes Raman Scattering), multiphoton imaging opens up the possibility of characterizing the cosmetic ingredients distribution inside the skin and founds application in other cosmetic domains such as hydration or antiperspirants. Developments in user-friendly, ultrasensitive, compact, multimodal imaging systems, on-the-fly data analysis and the synthesis of cosmetic ingredients with non-linear optical properties will certainly allow trespassing the todays frontiers of cosmetic applications.

We recently proposed a method for selective visualization of topical drug distribution within human facial skin using two-photon fluorescence lifetime imaging along with non-Euclidean phasor analysis as a pharmacokinetics and pharmacodynamics imaging toolkit. In order to improve the efficacy of topical drug delivery toward the treatment of inflammatory acne, we have now developed a combination topical gel containing both minocycline and a retinoid. Since both drugs have unique fluorescence lifetimes compared to skin, we were able to selectively visualize the distribution of minocycline and the retinoid within ex vivo human facial skin while isolating the contributions of the three components.

Aberrant metabolism mechanisms are a well-established hallmark of cancer. Exploiting tumor metabolism as a therapeutic target is being actively pursued. De novo synthesis of fatty acids by fatty acid synthase (FASN) is a particularly attractive mechanism to target because increased lipid synthesis is associated with more aggressive tumor. In particular, our work focuses on the reformulation of orlistat, an FDA-approved lipase inhibitor that also inhibits the thioesterase domain of FASN. We report on the rationale, synthesis, efficacy, delivery, and limitations of a novel nanoparticle formulation of orlistat in the goal of targeting the FASN pathway.

We used intravital multiphoton microscopy to study the recovery of hepatobiliary metabolism following carbon tetrachloride (CCl₄) induced hepatotoxicity in mouse. Our images were processed by a first order kinetic model to generate rate constant resolved images of the mouse liver. At Day 14 following induction, a restoration of the mouse hepatobiliary function was found. Our approach allows the study of the response of hepatic functions to chemical agents in real time and is useful for studying pharmacokinetics of drug molecules through optical microscopic imaging.

There is a great unmet need to non-invasively quantify the active versus passive delivery of drugs in preclinical studies. Quantifying probe-receptor interactions, or target engagement in living systems, is critical as it directly correlates with drug efficacy. We selected the transferrin receptor (TfR) as a target, since TfR is overexpressed in breast cancer cells. MFLI-FRET enables the quantification of transferrin (Tf) internalization into the cells by measuring FRET between receptor-bound Tf donor and acceptor near infrared (NIR) fluorophore pairs, based on the reduction of donor fluorophore lifetime in live intact mice. In this study we compared FRET levels in aggressively growing triple negative MDA-MB-231 breast cancer tumors to estrogen receptor positive T47D tumors, which are less dense and slowly growing. Unlike in T47D xenografts, in MDA-MB-231 tumors FRET donor fraction (FD%) was very low or undetectable in first few hours post injection. Only by 24-48 h p.i. FD% reached comparable to T47D FD% levels. Immunohistochemical analysis of excised tumors showed that TfR density levels were similar in both types of tumors. This suggests that ligand penetration inside the MDA-MB-231 tumors is impaired due to microenvironment features, such as the perfusion defects, elevated stroma stiffness and interstitial fluid pressure. We plan to measure functional blood flow using contrast-enhanced Doppler Ultrasound imaging in tumors to further validate MFLI-FRET data. Overall, MFLI-FRET is well suited for guiding the development of targeted drug therapy in preclinical studies as analytical tool to monitor and quantify drug penetration in heterogeneous breast cancer xenografts.

Oncology drug development is costly and ineffective with a 5% success rate from first-in-man studies to new drug registration. A task force set up to address issues in anticancer therapy development highlighted the necessity to demonstrate proof of mechanism and obtain information on key pharmacokinetic aspects of drug behaviour in both preclinical and early clinical trials. Spectroscopically bioorthogonal Raman detection in the “cellular-silent’ region presents an optimal region for drug imaging, as there is minimal contribution from endogenous cellular biomolecules thus improving detection sensitivity. This offer the potential to transform our understanding of intracellular drug distribution and mechanism of action. We demonstrate the utility of stimulated Raman scattering (SRS) in mapping intracellular concentrations of the tyrosine kinase inhibitor ponatinib used in the treatment of chronic myeloid leukemia and link this to resistance mechanisms associated with ponatinib treatment. As few drugs have inherent Raman active groups (5% of FDA approved drugs) further adoption and widespread use of SRS will require addition of bioorthogonal tags in the majority of cases. We have established an effective workflow for the design and use of Raman-labels for intracellular imaging. An iterative strategy of Raman-label design and validation identified minimally perturbative bis(alkyne) labels with approximately 60-fold increase in Raman scattering activity, compared to the mono-alkynes previously used. Combining SRS microscopy with this biorthogonal Raman labelling approach enabled direct visualisation of drug uptake to be correlated with markers of cell cycle status, and mapped across intracellular structures using multi-modal imaging platforms.

Quantification of protein concentrations is often a static and tissue destructive technique. Paired-agent imaging (PAI) using matched targeted and untargeted agents has been established as a dynamic method for quantifying the extracellular domain of epidermal growth factor receptor (EGFR) in vivo in a variety of tumor lines. Here we extend the PAI model to simultaneously quantify the extracellular and intracellular regions of EGFR using novel cell membrane permeable fluorescent small molecules, TRITC-erlotinib (targeted) and BODIPY-N-erlotinib (non-binding control isoform) synthesized in house. An EGFR overexpressing squamous cell carcinoma cell xenograft tumor, A431, was implanted on the chorioallantoic membrane (CAM) of the embryonated chicken egg. In total six fluorescent molecules were administered and monitored over 1 h using multi-spectral imaging. EGFR concentrations were determined using both extracellular and intracellular PAI methods. The fluorescent molecules used for extracellular PAI were ABY-029, an anti- EGFR Affibody molecule conjugated to IRDye 800CW, and a Control Imaging Agent Affibody molecule conjugated to IRDye 680RD. The intracellular PAI (iPAI) fluorescent molecules were cell membrane penetrating TRITC-erlotinib, BODIPY-N-erlotinb, and BODIPY TR carboxylate, as well as cell membrane impermeant control agent, Alexa Fluor 647 carboxylate. Results from simultaneous imaging of both the extracellular and intracellular binding domains of EGFR indicate that concentrations of intracellular EGFR are higher than extracellular. This is anticipated as EGFR exists in two distinct populations in cells, cell membrane bound and internalized, activated protein. iPAI is a promising new method for quantifying intracellular proteins in a rapid tumor model on the chicken CAM.

To successfully develop and manufacture a generic drug product, the latter is expected to be bioequivalent to its referencelisted drug, i.e. to show no significant difference in the rate and extent of absorption of the active pharmaceutical ingredient. Optical spectroscopy methods based on vibrational spectroscopy imaging have recently attracted significant attention as potentially viable approaches to quantify drugs’ pharmacokinetics in vivo. However, substantial hurdles still exist due to signal interference from surrounding tissues, significant attenuation of signal in the depth of tissue to optical absorption and scattering, and a lack of quantifiable ways of assessing the signal generated from a drug compound in the depth of a tissue. In this report, we evaluated the major challenges of quantification and sensitive and reproducible analysis of drug distribution in tissues using Raman spectroscopy. Specific attention is given to the noise assessment, which affects both the sensitivity and reproducibility of data.

Pre-clinical toxicology is a statutory requirement of drug development and plays a significant role in reducing attrition in drug discovery. Histopathology and indirect methods such as measurement of toxicity-associated systemic markers in blood or urine samples are the state-of-the-art techniques for toxicity evaluation. Further improvements over these conventional techniques are needed to detect signs of drug-induced toxicity at earlier stages with higher sensitivity and specificity. Multiphoton nonlinear imaging techniques such as two-/three-photon microscopy (2PF/3PF), fluorescence lifetime imaging microscopy (FLIM), second/third harmonic generation (SHG/THG) and coherent anti-Stokes Raman scattering (CARS) microscopy can extract complimentary structural and metabolic information of the target tissue in a label-free manner. In this study, we investigated the capability of a multimodal multiphoton microscopy technique (2PF/3PF/SHG/THG/FLIM/CARS) for detecting both functional and structural changes associated with drug-induced toxicity. Cisplatin, a platinum-based chemotherapy drug, is a cytotoxic agent used to treat many types of cancers. Common side effects of Cisplatin include nephrotoxicity and gonadal dysfunction. We obtained multimodal optical images of organs such as kidney, liver, and testis harvested from mice treated with a single dose of Cisplatin (3mg/kg) by intraperitoneal injection. A control group was treated with 0.9% saline. Structural and metabolic biomarkers related to Cisplatin-induced toxicity were identified and characterized from these multimodal optical images obtained ex vivo. The preliminary results suggest that it may be possible to develop a novel platform for drug toxicity identification and assessment based on multimodal nonlinear optical imaging techniques.

The high sensitivity and low cost of fluorescence imaging enables fluorescence molecular tomography (FMT) as a powerful noninvasive technique in applications of tracer distribution visualization. With the development of targeted fluorescence tracer, FMT has been widely used to localize the tumor. However, the visualization of probe distribution in tumor and surrounding region is still a challenge for FMT reconstruction. In this study, we proposed a novel nonlocal total variation (NLTV) regularization method, which is based on structure prior information. To build the NLTV regularization term, we consider the first order difference between the voxel and its four nearest neighbors. Furthermore, we assume that the variance of fluorescence intensity between any two voxels has a non-linear inverse correlation with their Gaussian distance. We adopted the Gaussian distance between two voxels as the weight of the first order difference. Meanwhile, the split Bregman method was applied to minimize the optimization problem. To evaluate the robustness and feasibility of our proposed method, we designed numerical simulation experiments and in vivo experiments of xenograft orthotopic glioma models. The ex vivo fluorescent images of cryoslicing specimens were regarded as gold standard of probe distribution in biological tissue. The results demonstrated that the proposed method could recover the morphology of the tracer distribution more accurately compared with fast iterated shrinkage (FIS) method, Split Bregman-resolved TV (SBRTV) regularization method and Gaussian weighted Laplace prior (GWLP) regularization method. These results demonstrate the potential of our method for in vivo visualization of tracer distribution in xenograft orthotopic glioma models.

Lithium is a key component in many anti-psychosis medications and can be transmitted to the breast-fed infants of medicated mothers. Using laser induced breakdown spectroscopy (LIBS), trace lithium levels are observed in the breast milk of lactating rats administered with lithium postpartum with limit of detection of LIBS about 0.1 ppm. Subsequently, the mammary glands of female rats were analyzed using LIBS and inductively coupled plasma mass spectrometry. Lithium at 1.06 μg/g concentration was measured in the mammary glands of lithium medicated subjects, but was below the limit of detection in controls. Lithium also increases iodine content (p<0.05), decreases phosphorus content, and increases the calcium/phosphorus ratio in the glands (p<0.05). Lithium is present in the breast milk and mammary glands of medicated female subjects and this is the likely route of entry to breast-fed infants. Lithium use in pre-partum has been associated with a number of negative effects in the newborn. These effects are conventionally resolved by reducing the pre-partum dose. However, current practice guidelines in post-partum discourage use of lithium during breast-feeding due to transient abnormalities of thyroid-stimulating hormone and blood urea nitrogen. This however, denies the infant the many benefits of breast feeding, or requires the mother to stop or change medications. The most recent guideline is still cautious as there is little evidence beyond a handful of case reports. Therefore, there is significant need for further research. This study helps establish the rat as an animal model for studying the biodistribution of lithium.