Application of human CFU-Mk assay to predict potential thrombocytotoxicity of drugs.

Megakaryocytopoiesis gives rise to platelets by proliferation and differentiation of lineage-specific progenitors, identified in vitro as Colony Forming Unit-Megakaryocytes (CFU-Mk). The aim of this study was to refine and optimize the in vitro Standard Operating Procedure (SOP) of the CFU-Mk assay for detecting drug-induced thrombocytopenia and to prevalidate a model for predicting the acute exposure levels that cause maximum tolerated decreases in the platelets count, based on the correlation with the maximal plasma concentrations (C max) in vivo. The assay was linear under the SOP conditions, and the in vitro endpoints (percentage of colonies growing) were reproducible within and across laboratories. The protocol performance phase was carried out testing 10 drugs (selected on the base of their recognised or potential in vivo haematotoxicity, according to the literature). Results showed that a relationship can be established between the maximal concentration in plasma (C max) and the in vitro concentrations that inhibited the 10-50-90 percent of colonies growth (ICs). When C max is lower than IC10, it is possible to predict that the chemicals have no direct toxicity effect on CFU-Mk and could not induce thrombocytopenia due to bone marrow damage. When the C max is higher than IC90 and/or IC50, thrombocytopenia can occur due to direct toxicity of chemicals on CFU-Mk progenitors.

[1]  D. Parent-Massin,et al.  Relevance of In Vitro Studies of Drug-Induced Agranulocytosis , 1993, Drug safety.

[2]  P. van Vlasselaer,et al.  Haemopoietic and osteogenic toxicity testing in vitro using murine bone marrow cultures. , 1995, Toxicology in vitro : an international journal published in association with BIBRA.

[3]  D. Parent-Massin,et al.  Prevalidation of a model for predicting acute neutropenia by colony forming unit granulocyte/macrophage (CFU-GM) assay. , 2001, Toxicology in vitro : an international journal published in association with BIBRA.

[4]  C. Pfaffenberger,et al.  Determination of organochlorine pesticides and metabolites in drinking water, human blood serum, and adipose tissue. , 1981, Journal of toxicology and environmental health.

[5]  Spielmann Horst,et al.  Practical Aspects of the Validation of Toxicity Test Procedures , 1995 .

[6]  Juan A. Bueren,et al.  The Use of In Vitro Systems for Evaluating Haematotoxicity , 1996 .

[7]  J. Sina,et al.  Usefulness of the in vitro bone marrow colony-forming assay in cellular toxicology , 1993 .

[8]  D. Parent-Massin,et al.  In vitro toxicity of trichothecenes on rat haematopoietic progenitors. , 1995, Food additives and contaminants.

[9]  I. Lewis,et al.  Standardization of the CFU-GM assay using hematopoietic growth factors. , 1996, Journal of hematotherapy.

[10]  Rodger Curren,et al.  The Validation of Toxicological Prediction Models , 1997 .

[11]  P. Miescher Drug-induced thrombocytopenia , 1973, Seminars in hematology.

[12]  Balls Michael,et al.  The Validation of Alternative Test Methods , 1996 .

[13]  D. Parent-Massin,et al.  Effects of a diphenyl-ether herbicide, oxyfluorfen, on human BFU-E/CFU-E development and haemoglobin synthesis , 1997, Human & experimental toxicology.

[14]  D Parent-Massin,et al.  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. , 2003, Toxicological sciences : an official journal of the Society of Toxicology.

[15]  H. Macdonald,et al.  Growth of mouse megakaryocyte colonies in vitro. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[16]  B. Naughton,et al.  Differential Effects of Drugs upon Hematopoiesis Can Be Assessed in Long-Term Bone Marrow Cultures Established on Nylon Screens , 1992, Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine.

[17]  W. Vainchenker,et al.  Megakaryocyte colony formation from human bone marrow precursors. , 1979, Blood.

[18]  J. Sobotka,et al.  A Review of Carbamazepine's Hematologic Reactions and Monitoring Recommendations , 1990, DICP : the annals of pharmacotherapy.

[19]  A. Angiolillo,et al.  A Phase I Clinical, Pharmacologic, and Biologic Study of Thrombopoietin and Granulocyte Colony-Stimulating Factor in Children Receiving Ifosfamide, Carboplatin, and Etoposide Chemotherapy for Recurrent or Refractory Solid Tumors: A Children's Oncology Group Experience , 2005, Clinical Cancer Research.

[20]  L. Gribaldo,et al.  Refinement of the colony-forming unit-megakaryocyte (CFU-MK) assay for its application to pharmaco-toxicological testing. , 2003, Toxicology in vitro : an international journal published in association with BIBRA.

[21]  R. Van Den Heuvel,et al.  Haematotoxicity testing in vitro using human cord blood haemopoietic cells. , 1997, Toxicology in vitro : an international journal published in association with BIBRA.

[22]  Michael Balls,et al.  The Role of Prevalidation in the Development, Validation and Acceptance of Alternative Methods , 1995 .

[23]  I. Dobo,et al.  Collagen matrix: an attractive alternative to agar and methylcellulose for the culture of hematopoietic progenitors in autologous transplantation products. , 1995, Journal of hematotherapy.

[24]  A. Kay Myelotoxicity of D-penicillamine. , 1979, Annals of the rheumatic diseases.

[25]  Robert D. Combes,et al.  Practical Aspects of the Validation of Toxicity Test Procedures , 1995 .

[26]  D. Parent-Massin,et al.  Improvement of megakaryocytic progenitor culture for toxicological investigations. , 2001, Toxicology in vitro : an international journal published in association with BIBRA.

[27]  D. Kerr,et al.  Can Pharmacokinetic Monitoring Improve Clinical Use of Fluorouracil? , 1999, Clinical pharmacokinetics.

[28]  L. Gribaldo,et al.  Role of SR-4987 stromal cells in the modulation of doxorubicin toxicity to in vitro granulocyte-macrophage progenitors (CFU-GM). , 1999, Life sciences.

[29]  B. Bannwarth,et al.  Haematological Adverse Effects of Histamine H2-Receptor Antagonists , 1988, Medical toxicology and adverse drug experience.

[30]  J B Houston,et al.  The Integrated Use of Alternative Methods in Toxicological Risk Evaluation , 1999, Alternatives to laboratory animals : ATLA.

[31]  A. Pessina The Granulocyte/Macrophage Colony-Forming Unit Assay , 1998 .

[32]  W. Bernstein,et al.  Mechanisms for Linezolid-Induced Anemia and Thrombocytopenia , 2003, The Annals of pharmacotherapy.

[33]  A P Worth,et al.  The Importance of the Prediction Model in the Validation of Alternative Tests , 2001, Alternatives to laboratory animals : ATLA.

[34]  M. Clynes Animal Cell Culture Techniques , 1998, Springer Lab Manual.