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.