A very successful field of in vitro toxicology is predicting reversible neutropenia caused by acute cytotoxic drug exposure. Recent success was built on research in 1950-1980 by experimental hematologists and cancer pharmacologists who discovered hematopoietic progenitors for the different myeloid blood cells; developed methods to grow progenitors as clonal colonies of morphologically recognizable blood cells and quantify the number of functional progenitors in a sample; demonstrated that these """"""""colony forming units"""""""" (CFUs) are targets of myelosuppressive cytoytoxic drugs in vivo and in vitro; and isolated and eventually cloned recombinant cytokines for completely defined and optimized cell culture media that stimiuated colony formation by progenitors from particular myeloid lineages. Our laboratory applied this knowledge successfully to develop in vitro hematotoxicology, i.e., predicting the nature of acute, reversible drug-induced neutropenia from the drug's in vitro toxicity to the CFUs. We published in vitro - clinical correlations for pyrazoloacridine and camptothecins. This research also discovered important principles and non-obvious maesurements to make that are critical for correctly predicting clinical outcome. Three in vitro prediction models for risk of severe neutropenia (CTC v2.0 Grade 3-4) have been published that define a population's risk of severe neutropenia as a function of dose or systemic exposure, rather than hazard classification (""""""""myelosuppressive or not'j. One model was selected as the subject of the current validation study by the European Centre for Validation of Altemative Methods (ECVAM), in which our laboratory is the North American participant. The hypotheses of this R21 application are (1) damage to gastrointestinal mucosa from acute cytotoxic exposure is conceptually identical to neutropenia and (2) the modeling and quality control concepts that made in vitro hematotoxicology successful will also be successful when applied to predict the acute dose/exposure that will cause reversible, severe mucosal damage (manifested clinically as stomatitis and mucosifs). GI progenitors (called """"""""transit cells"""""""") and stem cells, identified in mouse and rat, have been demonstrated to be a target of cytotoxic drugs and radiation. Mucosal populations can be grown in culture with retention of stem cell and progenitor function.
The Specific Aims of this R21 build on these discoveries and combine them with the assay development strategy used in hematotoxicology to develop an optimized in vitro clonogenic assay for """"""""CFU-Gl"""""""" progenitors that correctly predicts the relative risk of drug toxicity to GI mucosa versus CFU-GM. A subsequent R33 application will confirm similar drug exposure - CFU-GI survival curves in vitro and in vivo, perform a pre- validation study of the test, and adapt the test's configuration for application in the various stages of the drug development pathway with respect to through-put and cost.
