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The FMCA-GM assays, high throughput non-clonogenic alternatives to CFU-GM in preclinical hematotoxicity testing
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology. (Cancer Pharmacology and Informatics/Rolf Larsson)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology. (Cancer Pharmacology and Informatics/Rolf Larsson)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. (Division of Haematology)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
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2010 (English)In: Toxicology Letters, ISSN 0378-4274, E-ISSN 1879-3169, Vol. 194, no 3, 102-107 p.Article in journal (Refereed) Published
Abstract [en]

One of the most common dose limiting adverse effects in cancer treatment is myelotoxicity. The aim of this study was to develop an in vitro method for measuring potential myelotoxic properties of a drug candidate in a high throughput setting. Human CD34+ progenitor cells from umbilical cord blood were plated in 384-well microplates with drugs in liquid culture, supplemented with specific cytokines for the granulocytopoietic-macrophage lineage. After 7 or 14 days of proliferation and differentiation the cells were analyzed using the automated non-clonogenic fluorometric microculture cytotoxicity assay (FMCA). Two types of assays setups were evaluated, the FMCA-GM7 where cells were exposed to drugs directly after thawing and cytotoxicity measured on day 7 in contrast to the FMCA-GM14 where the cells were cultured 7 days prior to plating and drug exposure, with viability analysis on day 14 of differentiation. Drug sensitivity was similar in both assays and method validation was performed using 24 drugs with known myelotoxic profile (acyclovir, bortezomib, busulfan, carboplatin, chloramphenicol, chlorpromazine, cisplatin, cytarabine, clozapine, doxorubicin, erlotinib, etoposide, 5-fluorouracil, fludarabine, gefitinib, gemcitabine, hydroxyurea, imatinib, lomustine, melphalan, sorafenib, sunitinib, taxol and 6-thioguanine). The 50% inhibitory concentrations (IC50) from the FMCA-GM7 and the FMCA-GM14 correlated highly (r = 0.83) and (r = 0.82), respectively, with IC50 from the established clonogenic assay (CFU-GM), obtained from the literature. The current data suggests that the FMCA-GM could offer a simple and robust alternative to the CFU-GM assay in preclinical hematotoxicity studies.

Place, publisher, year, edition, pages
2010. Vol. 194, no 3, 102-107 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-124539DOI: 10.1016/j.toxlet.2010.02.006ISI: 000276933500008PubMedID: 20167269OAI: oai:DiVA.org:uu-124539DiVA: diva2:317654
Available from: 2010-05-04 Created: 2010-05-04 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Integrating Efficacy and Toxicity in Preclinical Anticancer Drug Development: Methods and Applications
Open this publication in new window or tab >>Integrating Efficacy and Toxicity in Preclinical Anticancer Drug Development: Methods and Applications
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Preclinical testing is an important part of cancer drug development. The aim of this thesis was to establish and evaluate preclinical in vitro methods useful in the development of new anticancer drugs.

In paper I, the development of non-clonogenic assays (FMCA-GM) using CD34+ stem cells for assessment of haematological toxicity was described. A high correlation was seen when comparing the 50% inhibitory concentrations (IC50) from FMCA-GM with the IC50 from the established clonogenic assay (CFU-GM).

In paper II, FMCA-GM was complemented with additional cell models, establishing a normal cell panel. In vitro toxicity towards the five normal cell types was compared with known clinical adverse event profiles. The normal cell panel roughly reflected the tissue specific toxicities but was most useful in the prediction of therapeutic index.

In paper III the use of peripheral blood lymphocytes from human, dog, rat and mouse to detect species differences in cellular drug sensitivity was described. Good agreement between our method and the established CFU-GM assay was observed.

In paper II the benefit of using primary tumour cells from patients to predict cancer diagnosis-specific activity was studied. The in vitro activity of fourteen anticancer drugs was tested in tumour samples of both haematological and solid tumour origin. In general, clinical activity was well reflected.

In paper IV, the efficacy and toxicity models were applied for experimental follow-up of a novel inhibitor of the ubiquitin-proteasome system, CB3 (Phosphoric acid, 2,3-dihydro-1,1-dioxido-3-thienyl diphenyl ester). In the preliminary characterization of CB3, antitumour activity and a favourable toxicity profile were displayed, although the exact mechanism of action remains to be elucidated. CB3 will therefore be further investigated.

In conclusion, the work presented here contributes to different parts of the preclinical drug development and the methods may aid in the characterization of anticancer compounds

Place, publisher, year, edition, pages
Uppsala: Acta Unversitatis Upsaliensis, 2011. 53 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 665
Keyword
Anticancer drugs, In vitro assays, Toxicity testing, Haematological toxicity, Primary tumour cells, Species difference
National Category
Pharmacology and Toxicology
Research subject
Clinical Pharmacology
Identifiers
urn:nbn:se:uu:diva-150361 (URN)978-91-554-8051-6 (ISBN)
Public defence
2011-05-12, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 13:15 (Swedish)
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Supervisors
Available from: 2011-04-19 Created: 2011-03-29 Last updated: 2011-05-05Bibliographically approved

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