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Issue 229 | December 2014 Biomarker Identification and Assay Development Biomarker assays are now an integral part of the drug discovery and development process, acting as indicators of drug efficacy, toxicity and disease progression, as well as assisting in patient selection and design of clinical trials1. In early stage drug discovery, biomarkers are used to validate in vitro target modulation and routine cellular screening. Typically, these assays are initiated in human cell lines, primary blood cells or isolated tissue cells. At this early stage, translational biomarkers are also investigated to identify markers of drug sensitivity that ultimately may be used in selection of patient populations. As projects progress, biomarker assays are developed for pharmacokinetic/ pharmacodynamic (PK/PD) models, profiling molecules prior to testing in longer term disease models as initial proof of concept. PK/PD models can also assist in dose-to-man scaling predictions for use in clinical trials. The biomarker assays developed during the in vitro discovery phases are frequently used as efficacy or toxicity endpoints in the clinic. Clinical trials, particularly in oncology, are frequently designed around these biomarkers. Charles River Early Discovery has broad expertise in biomarker identification and assay development across many therapeutic areas, including, oncology, CNS, and metabolic and respiratory diseases. We develop quantitative assays in primary or immortalized cells or disease tissue that can be used as pharmacodynamic or disease models. Ultimately, these models will act as efficacy and translational markers from the in vivo phase to the clinic. A broad range of endpoints can be employed to support biomarker selection, including genomic, proteomic, phosphoprotein and epigenetic markers. Assay technologies include Luminex® and Meso Scale for multiplexed endpoints, although antibody-driven FACS analysis and high-throughput mass spectrometry endpoints can also be applied to ensure that the most relevant and sensitive assays are employed. Charles River has considerable experience in the development of biomarker assays that have ultimately been used in the clinic. Our biomarker capabilities are catered to suit your needs, and are offered as a stand-alone service or a fully integrated drug discovery project. Below are just a few of the applications for Charles River’s biomarker identification and assay development services. Biomarker Identification The following illustrations demonstrate the breadth of Charles River’s biomarker identification and validation capabilities as applied in an oncology project. Using a validated tool compound, we were able to show significant changes to primary and secondary pathway phosphoproteins (Figure 1). This study was extended to look at modulation of specific genes known to be dependent on the phosphotarget (Figure 2) and validated in the same experiment by determining the gene signature elicited by short hairpin RNAs. The biomarker was successfully translated into mouse xenograft models with dose- and time-dependent inhibition of phosphoprotein observed in homogenized tumor material (Figure 3). Finally, the mechanism of action was confirmed by immunohistochemistry, with strong induction of apoptosis observed in tumor sections from animals who had received compound treatment for a set number of days (Figure 4). Figure 1. Western blotting of compound-treated cells showing dose-dependent decrease in phosphoprotein levels (A), increased secondary protein marker (B+C), induction of apoptosis marker (D), and protein loading control (E). To download previous Researcher issues on The SourceSM, please visit www.criver.com/thesource. Biomarker Assay Development Charles River Early Discovery has extensive experience in developing both efficacy and translational biomarker assays in cell lines, primary blood cells and tissues. Formats range from tissue mRNA levels, protein marker and signaling pathways to cellular phenotypic changes. Assays are commonly developed in immortalized cell lines initially, providing on-target cellular readouts to support medicinal chemistry optimization projects. Apoptosis % Inhibition % Inhibition Figure 2. Inhibition of gene expression in cell lines induced by tool compounds or shRNA These cellular assays can then be adapted for use in primary blood and tissue cells, including human cells obtained from an in-house donor panel, and from relevant species (typically rodents) for PD and disease models. The efficacy biomarker assays are used in PK/PD models to determine dosing regimens, and in disease models to correlate target coverage with disease-modifying effects. A range of assays for pharmacodynamic models have been developed from receptor occupancy in blood and tissue to functional readouts 125 of target function. showing inhibition 100 Nuclear Receptor Antagonist Biomarker Assays As part of125 a glucocorticoid receptor antagonist project, which 75 required compounds with good CNS penetration, ex vivo Vehicle 50 were developed Compound 100 binding assays in brain and other tissues to determine the extent and duration of antagonist binding.2 7525 The time course of antagonist binding in brain tissue, together with compound levels, was monitored to allow 50 0 PK/PD modelling (Figure 5). Data from this model were then 25 -25compounds for evaluation in a longer term used to select 1 10 100 1000 disease model, 0 and for functional effects in different regions of [Compound] (nM) the brain.2 -25 1 10 100 1000 [Compound] (nM) Apoptosis Figure 3. Pharmacodynamic inhibition of phosphoprotein by four tool compounds in mouse xenograft tumors. Protein levels were compared to vehicle-treated control to determine percentage inhibition of phosphoproteins. Figure 5. Antagonist nuclear receptor binding in rat brain at 2, 4 and 6 hours following a single oral dose of test compound. Compound Figure 4. Changes in apoptotic protein levels (stained brown) in mouse xenograft tumors after several days of treatment with tool compounds. 125 100 bition Vehicle 75 Tissue Biomarkers A lung phosphomarker was established to support an inhaled kinase inhibitor program to determine the inhibitor duration of action (Figure 8). The inhibitors showed slow dissociation kinetics from the target kinase, which gave prolonged pharmacodynamic properties despite rapid clearance of the compound from the lung. C on GPCR Antagonism Assay FACS analysis is one of the most versatile formats for biomarkers assays (Researcher issue 224, June 2014). A FACS whole blood leukocyte shape change assay was established as a biomarker assay to support a GPCR antagonist project. Antagonist effects were monitored as blockade of agonist-induced shape change (Figure 6). The whole blood assay was used in the PK/PD model to optimize the molecules, and ultimately in dose-to-man predictions prior to clinical trials. The PD assay was used in a Phase I trial to demonstrate inhibition of shape change at the predicted human dose. The Phase I shape change data was subsequently used to determine the dose range for Phase II proof of concept studies, and also to assist in patient selection for the trials. 125 75 Figure 8. Effect of a kinase inhibitor on lung phosphomarker levels in mice at various times following a single intratracheal dose. Peripheral Tissue Pharmacology 3000 14% ns Target Tissue Pharmacology 2000 3000 C on ed In du c nd ct iv ity ct iv ity nd In du c ct iv ity ct iv ity 0 nd nd om C + ed C on In du st itu tiv e ce d A 32% p<0.01 po u A ct iv ity ct iv ity 0 Tissue Target Pharmacology 1000 po u + C om A ed A ct iv ity In du ce d A st itu tiv e po u + C om A A ct iv ity C on In du c In du c ed C on In du ce d A Figure 9. Tissue-based biomarker measurement demonstrates separation of target and peripheral tissue pharmacology is achieved by topical compound dosing. st itu tiv e 14% ns 2000 Figure 7. Effect of a kinase inhibitor on phosphomarker levels in isolated human PBMCs and whole blood from the same donor. Pharmacology A ct iv ity 32% Peripheral p<0.01Tissue 1000 po u Blood Cell Biomarkers A phosphobiomarker assay was developed in isolated human peripheral blood mononuclear cells (PBMCs) and in whole blood to determine the impact of plasma protein binding on the activity of target kinase inhibitors (Figure 6). Initially, these assays were used to optimize the protein binding properties of inhibitors. Subsequently, the whole blood assay was used in pharmacodynamic models as an efficacy marker and in clinical studies as a measure of target engagement and efficacy. om Blood and Tissue Phosphomarker Assays C Figure 6. Inhibition of leukocyte shape change by a GPCR antagonist in human whole blood. + [Compound] (nM) 1000 ct iv ity 100 A 10 ce d 1 A ct iv ity -25 The key goal for the program in the following example was to maximize the therapeutic index by topical compound delivery to the target tissue with the aim of restricting peripheral pharmacological activity (and potential side-effect liability). A tissue-based biomarker assay was established that enabled parallel measurement of compound target engagement in both the target and peripheral tissue post topical compound dosing in mice. Using a functional pathway endpoint measurement, it was successfully established that topical compound dosing to the target tissue achieved selective pharmacological pathway modulation in the absence of peripheral pharmacological activity (Figure 9). ct iv ity 0 A 25 In du 50 st itu tiv e % Inhibition 100 Summary Charles River Early Discovery has broad expertise in biomarker identification that encompasses genomic, proteomic, epigenetic, phosphoprotein and phenotypic biomarkers across a range of cellular and tissue types. These assays have been successfully employed to demonstrate pharmacological target engagement, establish translational PK/PD relationships to support clinical dose projections and enable early clinical characterization of compound pharmacology for optimal dose setting for Phase II proof of concept studies. References 1. Colburn, W.A. Biomarkers in drug discovery and development: from target identification through drug marketing. J Clin Pharmacol. 43 (4), 329-41 (2003). 2. Zalachoras, I., Houtman, R., Atucha, E., Devos, R., Tijssen, A.M., Hu, P., Lockey, P.M., Datson, N.A., Belanoff, J.K., Lucassen, P.J., Joëls, M., de Kloet, E.R., Roozendaal, B., Hunt, H., Meijer, O.C. Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator. Proc Natl Acad Sci U S A., 110 (19), 7910-5 (2013). For additional information, please visit The SourceSM, a secure portal that provides registered users with direct access to the technical, scientific and educational resources available from Charles River. To register, visit www.criver.com/thesource. [email protected] www.criver.com