Validation of a flow cytometry based chemokine internalization assay for use in evaluating the pharmacodynamic response
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BioMed Central
Open Access
Methodology
Validation of a flow cytometry based chemokine internalization assay for use in evaluating the pharmacodynamic response to a receptor antagonist Timothy Wyant*1, Alan Lackey2,3 and Marie Green1 Address: 1Millennium Pharmaceuticals, Cambridge, MA, USA, 2Esoterix Center for Clinical Trials, Brentwood, TN, USA and 3Nodality Inc. Brentwood, TN, USA Email: Timothy Wyant* - [email protected]; Alan Lackey - [email protected]; Marie Green - [email protected] * Corresponding author
Published: 1 December 2008 Journal of Translational Medicine 2008, 6:76
doi:10.1186/1479-5876-6-76
Received: 3 September 2008 Accepted: 1 December 2008
This article is available from: http://www.translational-medicine.com/content/6/1/76 © 2008 Wyant et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract Pharmacodynamic assays are important in clinical trial design to investigate the relationship between drug concentration (pharmacokinetics) and drug "effect' or biological activity. Increasingly flow cytometry is being used to examine the pharmacodynamic effect of new drug entities. However, to date, the analytical validation of cytometry based assays is limited and there is no suitable guidance for method validation of flow cytometry-based pharmacodynamic assays. Here we report the validation of a flow cytometry-based chemokine internalization assay for use in evaluating the effect of a receptor antagonist in clinical trials. The assay method was validated by examining the stability of the reagent, assay robustness, sensitivity, repeatability and reproducibility precision. Experimental results show the assay reagent was stable over 26 weeks. The assay demonstrated a sensitivity to distinguish 0.005 μg/ml of a CCR2 antagonist with a %CV of 13.3%. The intra-assay repeatability was less than 15% with an inter-assay repeatability of less than 20%. In vivo study results demonstrated that the assay was consistent and a reliable measure of antagonist activity.
Background Chemokines are a class of small proteins that have potent chemotactic activity for cells of the immune system. In addition, they have the ability to activate leukocytes, to stimulate cytokine and proteolytic enzyme production, to mediate angiogenesis, and may be involved in cell proliferation and death. [1] The chemokine receptor CCR2 is widely expressed on mononuclear cells and a subset of memory (CD45RO+) CD4+ helper T cells. Activation of CCR2 by monocyte chemoattractant protein-1 (MCP-1), the major CCR2 ligand, is known to mediate chemotaxis and degranulation of monocytes as well as migration of activated effector memory T cells. [2,3] The MCP-1/CCR2
pathway has been implicated in a variety of disease states such as Rheumatoid Arthritis, Multiple Sclerosis, and Atherosclerosis
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