Prediction of bone marrow toxicity

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The likelihood that a compound will exhibit bone marrow toxicity in an in vivo assay predicted by the ability of the compound to inhibit at least eight kinases from a selected group.

This invention relates generally to the field of toxicology. More particularly, the invention relates to methods for predicting bone marrow toxicity, and methods for screening compounds for potential bone marrow toxicity.

Bone marrow ablation is often observed during in vivo toxicity studies for potent cytotoxic pharmaceutical compounds because progenitor bone marrow cells are highly proliferative and susceptible to cell cycle arrest, DNA damage, and apoptosis. Bone marrow toxicity is a major concern, particularly for drugs developed for indications other than oncology, as it can lead to neutropenia, anemia, and general immunosuppression. Thus, compounds that ablate bone marrow during in vivo toxicity studies are often dropped from further development, resulting in program delays and substantial financial expenditures.

Performing in vivo toxicological studies to determine bone marrow ablation is laborious, time consuming, expensive, and typically requires large quantities of compound. In vitro assays measuring specific progenitor stem-cell population toxicity and/or colony formation can be used as surrogates for in vivo toxicity studies, but these methods require further validation to address whether they can recapitulate the complexities and nuances observed with an in vivo study.

Kinases are enzymes responsible for phosphorylating substrates and disseminating inter- and intracellular signals. They fulfill integral roles in progenitor stem-cell differentiation as well as the initiation, propagation, and termination of mitosis in hematopoietic progenitor stem cells. Kinases are often the target of pharmaceutical research because many signaling cascades have known roles in a variety of diseases. Small molecule kinase inhibitors (SMKIs) often competitively bind to the kinase ATP binding pocket, blocking the ability of the enzyme to phosphorylate substrates. SMKIs often inhibit many kinases in addition to the desired target, due to the highly conserved nature of the ATP binding pocket within the kinome, thus toxicities associated with off-target kinase inhibition is a concern for this class of compounds. In particular, bone marrow toxicity or ablation, observed in the clinic or in in vivo toxicity studies, is a common toxicological liability for SMKIs because the kinases responsible for cellular differentiation or proliferation can be inhibited. We have now invented an in vitro method for predicting which compounds will demonstrate positive (i.e., bone marrow toxicity) results in in vivo bone marrow toxicity studies, using a method that is faster, uses smaller quantities of reagents, is easily automated, and is much cheaper. All publications cited in this disclosure are incorporated herein by reference in their entirety.

The invention provides a method for quickly determining the bone marrow toxicity in an in vitro toxicity assay by examining the interaction between the compound and a number of kinases(kinase binding and/or inhibition). As kinase inhibition and/or binding can be determined quickly, and by using automated methods, the method of the invention enables high-throughput screening of compounds for bone marrow toxicity (or lack thereof).

In one preferred embodiment, the inhibition of kinase activity is measured by determining the affinity of said compound for said kinase. In practice, binding and inhibition can be determined using methods known in the art. See, for example, M.A. Fabian et al., Nature Biotechnol (2005) 23:329-36, incorporated herein by reference in full. In general, the binding affinity of a compound for a given kinase correlates well with the ability of the compound to inhibit the activity of that kinase, so that binding affinity is a reliable substitute for inhibitory activity. Binding affinity may be determined by a variety of methods known in the art; for example by competitive assay using an immobilized kinase (or an immobilized test compound, or an immobilized competing ligand, any of which may be labeled). Compounds and kinases can be immobilized by standard methods, for example by biotinylation and capture on a streptavidin- coated substrate.