Application of the CFU-GM Assay to Predict Acute Drug-Induced Neutropenia: An International Blind Trial to Validate a Prediction Model for the Maximum Tolerated Dose (MTD) of Myelosuppressive Xenobiotics

Abstract

In a previous study of prevalidation, a standard operating procedure (SOP) for two independent in vitro tests (human and mouse) had been developed, to evaluate the potential hematotoxicity of xenobiotics from their direct and the adverse effects on granulocyte-macrophages (CFU-GM). A predictive model to calculate the human maximum tolerated dose (MTD) was set up, by adjusting a mouse-derived MTD for the differential interspecies sensitivity. In this paper, we describe an international blind trial designed to apply this model to the clinical neutropenia, by testing 20 drugs, including 14 antineoplastics (Cytosar-U, 5-Fluorouracil, Myleran, Thioguanine, Fludarabine, Bleomycin, Methotrexate, Gemcitabine, Carmustine, Etoposide, Teniposide, Cytoxan, Taxol, Adriamycin); two antivirals (Retrovir, Zovirax,); three drugs for other therapeutic indications (Cyclosporin, Thorazine, Indocin); and one pesticide (Lindane). The results confirmed that the SOP developed generates reproducible IC90 values with both human and murine GM-CFU. For 10 drugs (Adriamycin, Bleomycin, Etoposide, Fludarabine, 5-Fluorouracil, Myleran, Taxol, Teniposide, Thioguanine, and Thorazine), IC90 values were found within the range of the actual drug doses tested (defined as the actual IC90). For the other 10 drugs (Carmustine, Cyclosporin, Cytosar-U, Cytoxan, Gemcitabine, Indocin, Lindane, Methotrexate, Retrovir, and Zovirax) extrapolation on the regression curve out of the range of the actual doses tested was required to derive IC90 values (extrapolated IC90). The model correctly predicted the human MTD for 10 drugs out of 10 that had "actual IC90 values" and 7 drugs out of 10 for those having only an extrapolated IC90. Two of the incorrect predictions (Gemcitabine and Zovirax) were within 6-fold of the correct MTD, instead of the 4-fold range required by the model, whereas the prediction with Cytosar-U was approximately 10-fold in error. A possible explanation for the failure in the prediction of these three drugs, which are pyrimidine analogs, is discussed. We concluded that our model correctly predicted the human MTD for 20 drugs out of 23, since the other three drugs (Topotecan, PZA, and Flavopiridol) were tested in the prevalidation study. The high percentage of predicitivity (87%), as well as the reproducibility of the SOP testing, confirm that the model can be considered scientifically validated in this study, suggesting promising applications to other areas of research in developing validated hematotoxicological in vitro methods.

Targeting G-Quadruplex DNA Structures by EMICORON Has a Strong Antitumor Efficacy against Advanced Models of Human Colon Cancer

“…On the basis of these observations, it has been suggested that compounds with little differential in bone marrow sensitivity across species may have greater potential for reaching similar blood levels in patients as in mice (25). So, on the basis of the CFU-GM assay developed by Pessina and colleagues (46) and validated in several studies (26,47,48), we demonstrated that the mouse bone marrow is only about 5-fold less sensitive than human bone marrow, thus suggesting that EMICORON could reach a similar blood levels in humans as in mice. This is in line Real-time tumor dissemination was monitored by the imaging system.…”

Integration of in vivo and in vitro approaches to characterize the toxicity of Antalarmin, a corticotropin-releasing hormone receptor antagonist

“…Colony counts (normalized as a percentage of control) at each concentration were then plotted to generate the concentrationresponse curve. As previously reported (Erickson-Miller et al, 1997;Parchment et al, 1994Parchment et al, , 1998Pessina et al, 2003), the endpoint of the assay for quantifying inter-species differences in susceptibility to drug toxicity is the IC 90 , which is the concentration that inhibits colony formation by 90%. IC 90 values were calculated from log-linear regression on concentrationresponse between the flanking data points on either side of the 90% inhibition level.…”

Bone marrow CFU-GM and human tumor xenograft efficacy of three antitumor nucleoside analogs

“…Bone marrow is critically sensitive to nucleoside analogs (11). It is important to understand the toxicity of agents to bone marrow, and to determine whether bone marrow progenitor cells will have greater or less sensitivity to the agent than human malignant cells.…”

“…Bone marrow granulocytemacrophage-colony forming unit (CFU-GM) assays that compare the sensitivity of bone marrow cells across species are useful in predicting the blood levels of an agent that might be achieved in patients using blood levels tested in preclinical efficacy and safety studies as a guide. Mouse bone marrow is less sensitive to many cytotoxic agents than is human bone marrow, allowing blood levels in preclinical efficacy testing that can not be achieved in patients (11)(12)(13)(14)(15). An efficacious level of a compound with smaller or no differential in bone marrow progenitor sensitivity among species may have a better potential for reaching similar blood levels in patients as in mice.…”

“…If bone marrow toxicity is dose-limiting, these compounds may be more likely to be successful in reaching therapeutic doses. Pessina (11) suggested that through the use of the ratio of mouse/human CFU-GM IC 90 values and the mouse MTD that the human MTD of a compound could be predicted and thus the potential for achieving a therapeutic blood level in patients estimated.…”