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Integrating Efficacy and Toxicity in Preclinical Anticancer Drug Development: Methods and Applications
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology.
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 [en]
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: urn:nbn:se:uu:diva-150361ISBN: 978-91-554-8051-6OAI: oai:DiVA.org:uu-150361DiVA: diva2:407166
Public defence
2011-05-12, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 13:15 (Swedish)
Opponent
Supervisors
Available from: 2011-04-19 Created: 2011-03-29 Last updated: 2011-05-05Bibliographically approved
List of papers
1. The FMCA-GM assays, high throughput non-clonogenic alternatives to CFU-GM in preclinical hematotoxicity testing
Open this publication in new window or tab >>The FMCA-GM assays, high throughput non-clonogenic alternatives to CFU-GM in preclinical hematotoxicity testing
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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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-124539 (URN)10.1016/j.toxlet.2010.02.006 (DOI)000276933500008 ()20167269 (PubMedID)
Available from: 2010-05-04 Created: 2010-05-04 Last updated: 2012-03-14Bibliographically approved
2. In vitro evaluation of clinical activity and toxicity of anticancer drugs using tumor cells from patients and cells representing normal tissues
Open this publication in new window or tab >>In vitro evaluation of clinical activity and toxicity of anticancer drugs using tumor cells from patients and cells representing normal tissues
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2012 (English)In: Cancer Chemotherapy and Pharmacology, ISSN 0344-5704, E-ISSN 1432-0843, Vol. 69, no 3, 697-707 p.Article in journal (Refereed) Published
Abstract [en]

PURPOSE: The aim of this study was to evaluate a phenotypic cell panel with tumor cells from various patients and normal cells for preclinical profiles of antitumor efficacy and toxicity of anticancer drugs.

METHODS: The antitumor activity of fourteen anticancer drugs was tested in over one hundred tumor samples from patients with solid or hematological malignancies. Drug activity against four normal cell types was used for the assessment of normal tissue toxicity. In vitro activity of the drugs was compared with indications approved by the Food and Drug Administration and established adverse event profiles.

RESULTS: In general, in vitro drug activity in tumor cells from patients reflected known clinical activity of the drugs investigated. For example, the clinical activity of imatinib in chronic myeloid leukemia was clearly detected in the tumor panel. Further, and in accordance with clinical use, cisplatin and bortezomib showed high activity in ovarian cancer and myeloma samples, respectively. The normal cell models roughly reflected known clinical toxicity profiles and were able to detect differences in therapeutic index, e.g., between targeted drugs and classical cytotoxic agents. For example, the high tolerability of imatinib and the well-known renal toxicity of cisplatin were demonstrated.

CONCLUSIONS: In preclinical drug development, primary tumor cells from patients can be used for the prediction of cancer diagnosis-specific activity and may aid in the selection of diagnoses for clinical trials. By using tumor and toxicity panels together, information about therapeutic index may be derived, which may be useful when choosing among drug candidates with similar tumor effects.

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-150354 (URN)10.1007/s00280-011-1746-1 (DOI)000302325600013 ()21984220 (PubMedID)
Available from: 2011-03-29 Created: 2011-03-29 Last updated: 2014-10-27Bibliographically approved
3. Primary lymphocytes as predictors for species differences in cytotoxic drug sensitivity
Open this publication in new window or tab >>Primary lymphocytes as predictors for species differences in cytotoxic drug sensitivity
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2007 (English)In: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 21, no 6, 1174-1181 p.Article in journal (Refereed) Published
Abstract [en]

Several in vitro methods have been suggested to predict drug-induced haematotoxicity and species differences; the most commonly used being the clonogenic CFU-GM assay. The aim of the current study was to evaluate whether primary lymphocytes from peripheral blood, assayed with a short-term non-clonogenic assay, could be used to detect species differences in drug sensitivity, and offer an alternative to the CFU-GM assay. The effect of 17 different cytotoxic drugs on lymphocytes from human, dog, rat and mouse was evaluated. A higher sensitivity of human than mouse lymphocytes was seen for topotecan and for 3 of 5 antimetabolites tested. Clear species specificity was also seen for the proteasome inhibitor bortezomib where rodent cells were 50–300 times less sensitive than human cells. Good agreement between our data and published CFU-GM data was observed, suggesting that primary lymphocytes may be a useful model for species difference screening in drug development.

Keyword
Animals, Antineoplastic Agents/*toxicity, Cell Survival/drug effects, Cells; Cultured, Dogs, Female, Fluorometry, Humans, Leukocytes; Mononuclear/*drug effects, Male, Mice, Mice; Inbred BALB C, Rats, Rats; Inbred Strains, Species Specificity, Toxicity Tests/methods
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-15582 (URN)10.1016/j.tiv.2007.03.009 (DOI)000249542300022 ()17481850 (PubMedID)
Available from: 2008-02-21 Created: 2008-02-21 Last updated: 2011-05-05Bibliographically approved
4. Identification of CB3, a novel inhibitor of the ubiquitin-proteasome system
Open this publication in new window or tab >>Identification of CB3, a novel inhibitor of the ubiquitin-proteasome system
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(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:uu:diva-150349 (URN)
Available from: 2011-03-29 Created: 2011-03-29 Last updated: 2011-05-05

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