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Tutorials : Feb 15, 2010 (Vol. 30, No. 4)

Predicting Drug-Induced Myelotoxicity

CFC Assays Using Primary Bone Marrow Cells Show Utility in Toxicity Screening
  • Emer Clarke
  • ,
  • Eric Atkinson

Potential pharmaceuticals are typically screened for toxicity with high-throughput assays that utilize cultured cell lines and simple endpoints like cell death or inhibition of cell proliferation. Although these methods have allowed the screening of huge numbers of compounds with millions of data points, many of the lead compounds screened by such practices have experienced late drug failure due to unacceptable toxicity in the clinic.

One way to improve the efficiency and economics of the drug pipeline is to introduce a highly relevant/highly informative assay early in the drug discovery process that overcomes these limitations.

Primary cells have much more fastidious growth requirements and are often more sensitive to toxic agents than long-term cell lines. Relevant functional assays using primary cells from particular tissues are also often more predictive of actual toxic effects on the given tissues and organs in vivo than assays using cultured cell lines. Bone marrow represents an important target tissue for toxicity screening from which significant quantities of individual primary cells are relatively easily obtained and for which highly relevant, validated functional assays are available.

ReachBio is focused on highly predictive assays for compound-induced myelotoxicity using primary cells from bone marrow. The colony forming cell (CFC) assay using primary bone marrow cells has been used to predict for neutropenia (reduction of granulocytes), severe anemia (reduction of red blood cells), or thrombocytopenia (reduction of platelets) for a number of different compound classes including chemotherapeutic agents, antivirals, and immunosuppressive compounds.

The bone marrow cells containing progenitors of the various blood cell lineages are cultured in a semisolid matrix (for example, ReachBio’s ColonyGEL™) with appropriate cytokines to induce the progenitor cells to divide and differentiate into morphologically distinct colonies of mature cell types. The addition of test molecules to this system can be used to detect compound-induced changes in both number of colonies formed (quantitative) or colony size and morphology (qualitative), both of which infer toxicity (Figure 1).

By using combinations of cytokines that allow the simultaneous growth of both myeloid (granulocytic) and erythroid (red blood) cell colonies, it can be determined if a compound’s toxicity is restricted to a specific lineage or if the damage is more generalized. Another significant advantage of this assay system is that it allows for the simultaneous testing of multiple compounds, and analyses of colony numbers can predict whether compounds in combination will have additive or synergistic toxicity to the bone marrow compartment.

This may be of particular importance when developing new therapeutics for patients who are likely to have been heavily pretreated with traditional chemotherapeutic compounds (unintentional combination therapy) or new therapeutics that are designed to be used with other compounds to enhance the efficacy of both (intentional combination therapy).

Although many animal models (in vitro and in vivo) are useful tools for toxicity assessment, studies have revealed significant differences between human, dog, rat, and mouse hematopoietic progenitor cells with regard to their sensitivities to certain pharmaceuticals.

In particular, there are some classes of compounds where a curative dose (blood levels) in mice against a human tumor xenograft may not be achievable in patients due to a higher sensitivity of normal bone marrow cells in humans than in mice. For these reasons, some groups have looked at ratios of the human and mouse values for myeloid progenitor (CFU-GM) growth for specific drug classes (the camptothecins, for example).

The European Centre for the Validation of Alternative Methods recently validated the CFU-GM assay and suggested that through the use of the ratio between mouse and human IC90 CFU-GM values as well as the maximum tolerated dose (MTD) of the compound in mice, the MTD in patients could be predicted.

Kinase Inhibitors

While there has been significant previous experience in using CFC assays to evaluate the toxic effects of more traditional classes of therapeutics, little was known until recently about this assay’s utility in evaluating the newer classes of targeted therapeutics such as kinase inhibitors (KIs).

The success of Imatinib in targeting the ABL tyrosine kinase in chronic myeloid leukemia has prompted the development of a number of other KIs for the treatment of various cancers, making this drug class currently one of the most active areas in pharmaceutical development. Unfortunately myelotoxicity, in particular, neutropenia is often a major side effect of this compound class.

Using in vitro CFU-GM assays, scientists at ReachBio determined the IC50 values of six KIs (Imatinib, Lapatinib, Erlotinib, Dasatinib, Sorafenib, and Sunitinib) and compared them to the degree of clinical neutropenia caused by these drugs, as reported in the literature.

For this assay, functionally prequalified human bone marrow cell samples from three different donors were mixed with the KIs over a broad concentration range in ColonyGEL methylcellulose-based media, plated in 35 mm dishes (n=3), and the resulting CFU-GM colonies were enumerated. The range of IC50 values thus determined allowed the ranking of these compounds in terms of toxicity to the bone marrow progenitors.

As shown in Figure 2, there was a direct correlation between the reported clinical neutropenia and the IC50 values derived from the in vitro CFC assays, with lower IC50 values associated with increased neutropenia. This high degree of correlation between in vitro IC50 values and clinical neutropenia is the basis of ReachBio’s new HemoRANK™ assay system, which allows predictive myelotoxic potential ranking of a compound compared to other members of the same chemical class for which clinical toxicity data is already available, or between various analogues of a particular compound.

The data derived from the HemoRANK system can be used to compare structure-related toxicity information with structure-related activity information early in the development process and help guide decisions regarding which analogues to explore further or abandon. This type of information will be increasingly important since the success of KIs in the oncology field has driven the evaluation of these compounds for their utility in treating inflammation and other immune system diseases.

Since the life-threatening potential of these types of diseases is generally lower than that of cancer, the risk/benefit equation changes and the degree of acceptable toxicity is necessarily higher. There will, therefore, likely be increased pressure on companies developing KIs for nononcology applications to produce compounds with lower levels of myelotoxicity than previously associated with this compound class.

The ability of the CFU-GM assay and the HemoRANK system to accurately predict the myelotoxic potential of new KIs and rank the potential clinical neutropenia against the degree of neutropenia already established for marketed drugs of the same class therefore represents a useful and timely tool for companies developing new KIs for oncology and nononcology applications alike.

Although not yet a fully automated system, when performed by skilled and experienced personnel, these assays are reproducible and can be used to screen a large number of compounds early on in the drug discovery process.