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Non-perturbative imaging of intracellular drug exposure and drug response of kinase inhibitors

激酶抑制剂细胞内药物暴露和药物反应的非微扰成像

基本信息

批准号：
10606525
负责人：
Dan Fu
金额：
$ 38.5万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10606525
关键词：
3-Dimensional Acceleration Address Biological Assay Cells Chemicals Data Deuterium Disease Dorsal Drug Compounding Drug Exposure Drug Kinetics Drug Screening Drug Transport Drug resistance Environment Enzymes Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Fourier Transform Genetic Goals Growth Heterogeneity Image Imaging Techniques In Vitro Label Malignant Neoplasms Measurement Measures Methods Microscopy Modeling Molecular Monitor Penetration Pharmaceutical Preparations Pharmacotherapy Phenotype Physiologic pulse Play Population Proliferating Property Proteins Resolution Role Signal Transduction Techniques Technology Time Tissues Toxic effect Work cancer cell cell growth drug development drug discovery drug efficacy drug response prediction empowerment improved in vivo inhibitor therapy innovation innovative technologies inter-individual variation kinase inhibitor mouse model novel optical imaging precision drugs public health relevance rate of change response small molecule success theories therapeutic development tumor

项目摘要

Abstract: The past two decades have witnessed unparalleled success in the development of therapeutic kinase inhibitors targeting protein enzymes that are essential for cellular signaling cascades. The major challenges in kinase inhibitor drug treatment are interindividual variability, compromised drug efficacy, inevitable drug resistance, and toxicity. All of these properties depend strongly on intracellular drug concentration, which can be profoundly influenced by heterogeneous tissue penetration, drug transport, and lysosomal drug sequestration. However, currently there is no technology that can quantitatively examine intracellular concentration of kinase inhibitor drugs in living cells with subcellular spatial resolution. Stimulated Raman scattering (SRS) microscopy is an emerging chemical imaging technique that monitors molecule-specific vibrational signatures to provide quantitative, spatially resolved measurements of molecular concentration. We propose to develop novel SRS- based methods to enable non-perturbative, quantitative determination of single cell drug exposure for the first time. The first method uses the pH partition theory to derive cytosolic drug concentration based on lysosomal drug sequestration of weakly basic drugs. The second method uses an ultrasensitive Fourier-transform SRS technique and advanced chemometric analysis to directly determine cytosolic drug concentration. We will use these innovative methods to determine EGFR inhibitor penetration and drug sequestration in vitro using 3D tumor spheroids and in vivo using the dorsal skinfold chamber mouse model. In addition, we will systematically vary the physicochemical properties of drug compounds and determine their influence on drug transport, sequestration, and penetration. The second goal of this proposal is to elucidate the heterogeneity of cell response to kinase inhibitor drug treatment. Drug response of cancer cells depend on not only their genetic aberrations, but also their phenotypic states and microenvironments. Traditional proliferation assays measure the ensemble response of a cell population and are unable to resolve the highly heterogeneous drug response of cells in a 3D environment. We propose to develop a quantitative, high sensitivity single-cell growth-rate measurement technique based on deuterium pulse labeling. We will validate the use of growth rate change as an accurate predictor of drug response. By combining single cell drug exposure and drug response measurements in 3D tumor spheroids, we will further dissect the influence of drug penetration, intracellular drug exposure, and cell microenvironment on cell drug response. The proposed work builds on our strong expertise in label-free optical imaging and addresses key challenges in drug discovery and development by providing unprecedented measurement capabilities. The technologies and methods developed can be broadly applied to small molecule drugs, with great potentials to accelerate early stage drug discovery and empower personalized drug screening.

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