Toxicology Letters 188 (2009) 98–103 Contents lists available at ScienceDirect Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet In vitro to in vivo concordance of a high throughput assay of bone marrow toxicity across a diverse set of drug candidates Andrew J. Olaharski a,∗ , Hirdesh Uppal a , Matthew Cooper a , Stefan Platz a , Tanja S. Zabka b , Kyle L. Kolaja a a Discovery and Investigative Safety, Non Clinical Safety, Roche Palo Alto, 3431 Hillview, Palo Alto, CA 94304, United States b Pathology, Non Clinical Safety, Roche Palo Alto, 3431 Hillview, Palo Alto, CA 94304, United States article info Article history: Received 17 January 2009 Received in revised form 27 February 2009 Accepted 13 March 2009 Available online 25 March 2009 Keywords: Hematopoietic toxicity Bone marrow toxicity CFC-GEMM abstract The development of predictive toxicology assays is necessary to optimize the drug candidate selection process. The colony forming assay (CFA) is used routinely to assess bone marrow toxicity and represents a viable tool for the discovery toxicologist, but the assay is not widely accepted as a standard screening tool due to technical challenges. A higher throughput and standardized version of the assay recently was developed such that the proliferative capacity of a cell lineage is measured indirectly via ATP levels, replacing the cumbersome identification and enumeration of specific colonies. In this study, a high- throughput assay of bone marrow toxicity prediction using the granulocyte, erythrocyte, monocyte, and macrophage (GEMM) progenitor cell lineage was evaluated using a training set of 56 structurally diverse compounds with known in vivo bone marrow effects. In general, compounds identified as toxic in vivo had lower IC 50 values, whereas those identified as non-toxic had higher IC 50 values. Concordance (i.e., predictive accuracy) to in vivo bone marrow toxicity results was 82% when an in vitro toxicity threshold of 20 M was used. Additional experiments in other hematopoietic lineages were conducted to determine if predictivity of several false positive and negative compounds in the GEMM lineage could be improved; however an increase in sensitivity or specificity was not observed. The high-throughput GEMM assay has good concordance to in vivo bone marrow toxicity results and, with the high-throughput and standardized format, can be incorporated readily into the pharmaceutical toxicological screening paradigm, aiding in the early identification of compounds that eventually may fail due to bone marrow toxicity. © 2009 Elsevier Ireland Ltd. All rights reserved. 1. Introduction A persistent challenge in discovery toxicology is identifying in vitro assays that can predict in vivo toxicity. Accurate assessment of potential toxicological risk using in vitro models is limited by a number of factors that can affect whether a toxicological conse- quence of a compound will be manifested in an in vitro assay, such as effective metabolism, concentration to exposure extrapolation, altered cellular architecture, and lack of multiple cell types. Thus, a predominant hypothesis to increase the predictive accuracy of an in vitro assay is to increase its overall in vivo “likeness”. Advances in optimized cell culture conditions and improved biological under- standing of stem and progenitor cells provide novel opportunities to mimic in vivo processes. These improvements have expanded the mechanistic and investigative tool set of the discovery toxicologist (Cezar, 2007). Hematopoiesis, the process of creating different cell lineages that comprise the cell types found in blood, relies on differen- ∗ Corresponding author. E-mail address: andrew.olaharski@roche.com (A.J. Olaharski). tiation from a pluripotent stem cell into several progenitor cell populations and subsequent terminal differentiation into the blood cell populations. Initial efforts to reproduce hematopoiesis in vitro began in the 1960s when colony-forming assays (CFA) first were developed (Bradley and Metcalf, 1966; Pluznik and Sachs, 1966). Specific cytokines and growth factors direct the primitive lympho- hematopoieitic stem cells into different cell lineages, forming lineage-specific colonies that are enumerated in the CFA. This work laid the foundation for the current understanding of the lympho- hematopoietic lineage hierarchy (Rich and Hall, 2005), which is arguably the most investigated and understood cellular differen- tiation cascade (Orkin and Zon, 2008). This knowledge enables the selective growth of a particular lineage of hematopoetic cells while simultaneously suppressing the growth of others, and thus permits the identification of xenobiotic-mediated bone marrow toxicities to isolated primitive precursor, progenitor, and/or stem cell pop- ulations in vitro (Lan et al., 2004; Gribaldo et al., 2000). The CFA assay demonstrates a high concordance between the in vitro and in vivo results, albeit for a small number of hematopoietic toxi- cants (Rich and Hall, 2005; Pessina et al., 2003), providing proof of concept that the approach may be of benefit for toxicological testing. 0378-4274/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2009.03.012