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Preclinical methods for the identification and characterization of molecules for development into new cancer drugs were investigated. Based on repurposing, i.e. the exploration of currently prescribed drugs for new indications, and as a result of a new high throughput screening (HTS) approach, the benzimidazoles benomyl and carbendazim, the alkaloid cryptolepine and the acridine acriflavine were found interesting to characterize using these methods. In mice the benzimidazoles inhibited 3H-thymidine incorporation in tissues with high cell renewal, with benomyl being more active than carbendazim.  They were rapidly absorbed with highest amounts seen in the liver, kidneys and gastro-intestinal lumen as evidenced from distribution of 14C-labeled drugs. In human tumour cell lines, the benzimidazoles showed a similar activity pattern but benomyl was more potent. This was true also in tumour cells from patients but carbendazim was relatively more active against solid tumours. Analyses of drug activity cross-resistance patterns and of drug activity – gene expression correlations in a cell line panel suggested multiple mechanisms of action for the benzimidazoles. Cryptolepine was widely distributed to tissues in vivo in the mice. It was more potent than the benzimidazoles in tumour cells, with highest activity in haematological malignancies but some patient samples of breast, colon and non small-cell lung cancer were sensitive. Cross-resistance analysis indicated cryptolepine to be a topoisomerase II inhibitor whereas drug activity – gene expression correlations suggested additional mechanisms of action. HTS on 2 000 molecules in colon cancer cell lines and normal cells identified acriflavine as a hit molecule, subsequently shown to have unprecedented activity against colorectal cancer tumour cells in patient tumour samples. Connectivity map analysis, based on drug induced gene expression perturbation patterns in a tumour cell line, indicated acriflavine to be a topoisomerase inhibitor, subsequently confirmed in a plasmid relaxation assay. In conclusion, repurposing of drugs and HTS using stringent activity criteria followed by preclinical characterization might contribute to more efficient development of new cancer drugs.

The aims of this thesis were to investigate the utility of in vitro drug sensitivity testing to optimize the use of cancer chemotherapy and to assess the properties of a new cancer drug in a phase I clinical trial. Tumour cells from patients were analysed with the short-term Fluorometric Microculture Cytotoxicity Assay (FMCA). In samples from a wide spectrum of tumour types, the effect of the drug combination FEC (5Fu-epirubicin-cyclophosphamide) was generally appropriately predicted from the effect of the best component drug. However, of samples intermediately sensitive to the best single drug, 45% converted to sensitive when testing the combination. Thus, combination testing may identify advantageous interactions and improve in vitro test performance. In tumour samples from peritoneal carcinomatosis, significant differences in drug sensitivity between diagnoses were observed, cross-resistance between most drugs was modest or absent, and the concentration-effect relationships for two drugs in individual samples varied considerably. Thus, for optimal selection of drugs for intraperitoneal chemotherapy, differences in drug sensitivity at the diagnosis and individual patient level should be considered. In samples from patients with ovarian carcinoma, drug sensitivity was related to tumour grade, histologic subtype and patient treatment status. In a homogeneous subset of patients, the FMCA predicted individual patient tumour response with high sensitivity and specificity. Thus, if carefully interpreted in the context of important clinical variables, in vitro testing could be of value for individualizing chemotherapy in ovarian cancer. Employing a once weekly dosing schedule in a phase I trial, the mechanistically new and preclinically promising NAD depleting drug CHS 828 produced dose limiting thrombocytopenia and gastrointestinal toxicity without clear evidence of anti-tumour efficacy. It is concluded that in vitro drug sensitivity testing could be a way to optimize the use of chemotherapy and that successful development of new cancer drugs needs improved strategies.

<p>This thesis describes the use of a new technology, the Cytosensor^® microphysiometer, in the in vitro evaluation of cytotoxic drugs, using the lymphoma cell line U-937 GTB and primary cultures of tumour cells from patients as model systems. The method was specifically applied to study the metabolic effects of the novel cyanoguanidine N-(6-(4-chlorophenoxy)hexyl)-N’-cyano-N’’-4-pyridylguanidine, CHS 828, currently in phase I/II clinical trials. </p><p>The Cytosensor^® measures metabolic effects as changes in the rate of extracellular acidification of cells exposed to a drug by perfusion. A number of standard cytotoxic drugs were found to produce typical and reproducible acidification response patterns during observation times up to 20 h. There seemed to be a relationship between a decrease in acidification and cytotoxicity, measured in the fluorometric microculture cytotoxicity assay (FMCA), after 20-24 h of continuous drug exposure.</p><p>In U-937 cells, CHS 828 induced a cytotoxic effect characterised by a steep concentration-response relationship followed by a plateau. After 24 h of incubation the DNA and protein synthesis were turned off. CHS 828 was found to produce a rapid and prolonged increase in extracellular acidification and lactate production similar to that of the structurally related mitochondrial inhibitor m-iodobenzylguanidine (MIBG). The CHS 828 induced acidification was observed in cell lines as well as in cells from various tumour types from patients and probably originates from increased glycolytic flux. The effects may be secondary to block of oxidative phosphorylation in the mitochondria, but the relevance of the early acidification is not clear. CHS 828 seemed to induce a late, at approximately 15 h, inhibition of the glycolysis followed by loss of ATP and subsequent cell death. After exposure to MIBG the loss of ATP and cell death occurred earlier and in parallel. The effects of CHS 828 were not found to resemble those of the structurally related polyamine biosynthesis inhibitor methylglyoxal-bis(guanyl-hydrazone) (MGBG). Thus, CHS 828 may represent a new and, thus, interesting mode of cytotoxic action worthwhile for further development.</p><p>In combinatory studies, a synergistic interaction was demonstrated between CHS 828 and the non-toxic drug amiloride. Additive-to-synergistic effects were also seen between CHS 828 and the bioreductive cytotoxic drug mitomycin C. In U-937 cells as well as in tumour cells from patients, CHS 828 demonstrated synergistic interactions in combination with melphalan and etoposide. </p><p>It is concluded that measurement in the Cytosensor^® microphysiometer of early cellular metabolic changes is a feasible and potentially valuable complement to more conventional methods used in the evaluation of anticancer agents. </p>

This thesis describes the use of a new technology, the Cytosensor® microphysiometer, in the in vitro evaluation of cytotoxic drugs, using the lymphoma cell line U-937 GTB and primary cultures of tumour cells from patients as model systems. The method was specifically applied to study the metabolic effects of the novel cyanoguanidine N-(6-(4-chlorophenoxy)hexyl)-N’-cyano-N’’-4-pyridylguanidine, CHS 828, currently in phase I/II clinical trials. The Cytosensor® measures metabolic effects as changes in the rate of extracellular acidification of cells exposed to a drug by perfusion. A number of standard cytotoxic drugs were found to produce typical and reproducible acidification response patterns during observation times up to 20 h. There seemed to be a relationship between a decrease in acidification and cytotoxicity, measured in the fluorometric microculture cytotoxicity assay (FMCA), after 20-24 h of continuous drug exposure. In U-937 cells, CHS 828 induced a cytotoxic effect characterised by a steep concentration-response relationship followed by a plateau. After 24 h of incubation the DNA and protein synthesis were turned off. CHS 828 was found to produce a rapid and prolonged increase in extracellular acidification and lactate production similar to that of the structurally related mitochondrial inhibitor m-iodobenzylguanidine (MIBG). The CHS 828 induced acidification was observed in cell lines as well as in cells from various tumour types from patients and probably originates from increased glycolytic flux. The effects may be secondary to block of oxidative phosphorylation in the mitochondria, but the relevance of the early acidification is not clear. CHS 828 seemed to induce a late, at approximately 15 h, inhibition of the glycolysis followed by loss of ATP and subsequent cell death. After exposure to MIBG the loss of ATP and cell death occurred earlier and in parallel. The effects of CHS 828 were not found to resemble those of the structurally related polyamine biosynthesis inhibitor methylglyoxal-bis(guanyl-hydrazone) (MGBG). Thus, CHS 828 may represent a new and, thus, interesting mode of cytotoxic action worthwhile for further development. In combinatory studies, a synergistic interaction was demonstrated between CHS 828 and the non-toxic drug amiloride. Additive-to-synergistic effects were also seen between CHS 828 and the bioreductive cytotoxic drug mitomycin C. In U-937 cells as well as in tumour cells from patients, CHS 828 demonstrated synergistic interactions in combination with melphalan and etoposide. It is concluded that measurement in the Cytosensor® microphysiometer of early cellular metabolic changes is a feasible and potentially valuable complement to more conventional methods used in the evaluation of anticancer agents.

Background: Cancer is the second leading cause of death in economically developed countries. The use and availability of oral treatment for cancer has increased dramatically in the past 10 years. Few studies have described the use of oral chemotherapy in non-traditional ambulatory settings by health care professionals across different specialties. Objective: The purpose of this study is to describe the usage of oral chemotherapeutic medications in ambulatory settings. Methods: Cross sectional study of 2007 NAMCS Survey analysis involving 21,761 subjects aged 18 years and above with cancer who participated in the 2007 National Ambulatory Medical Survey (NAMCS). Main Outcome Measure: Physician-reported use of oral chemotherapeutic medications (includes all major drug classes) as indicated on questionnaire for 2007 NAMCS survey. Results: Health care providers in non-traditional settings are less likely to prescribe oral chemotherapy than in traditional ambulatory settings (Adjusted odds ratio (AOR)=0.65{95% confidence interval: 0.61-0.69}). The study results suggest that oncologists are prescribing oral anti-cancer drugs the most as compared to other physician specialties. Conclusion: Health care providers in non-traditional settings are less likely to prescribe oral chemotherapy than in traditional ambulatory settings. Primary care physicians may have limited experience in monitoring and prescribing these potentially toxic medications. Clear guidelines are required for the use of oral chemotherapy medications, considering the potential for their use in non-traditional ambulatory settings and by non-oncologists.

<p>Despite major advances in the understanding of the biology of chronic lymphocytic leukaemia (CLL), progress in improving its treatment has been limited and it still remains an incurable disorder. In the present research, we have performed <i>in vitro</i> drug sensitivity testing of primary CLL cells for preclinical evaluation of cytotoxic drugs, using the fluorometric microculture cytotoxicity assay (FMCA).</p><p>The tumour type-specific activities of 14 standard drugs, evaluated <i>in vitro</i> on tumour cells from patients with CLL and acute leukaemias, were in good agreement with their known clinical activities. A correlation between drug treatment and development of cellular drug resistance was demonstrated in CLL, but not in the acute leukaemias. Moreover, the nucleoside analogues fludarabine, cladribine, cytarabine and gemcitabine, as well as the anthracycline idarubicin, were highly active in CLL cells.</p><p>A new cytotoxic drug candidate, CHS 828, was evaluated in primary cell cultures from a broad spectrum of tumours. CHS 828 was highly active against haematological malignancies <i>in vitro</i>, especially CLL, but also against some solid tumours. The drug appeared to be non cross-resistant with standard drugs.</p><p>In addition, the relationship between drug sensitivity <i>in vitro</i> and a recently described prognostic factor in CLL, the mutational status of the immunoglobulin variable heavy chain (IgV_H) gene, was evaluated. Interestingly, cells with unmutated IgV_H genes were more chemosensitive than the mutated cells. </p><p>In summary, our results indicate that <i>in vitro</i> studies on tumour cellsfrom leukaemia patients may yield considerable information regarding the activity, mechanisms of action and cross-resistance of cytotoxic drugs, as well as concerning the relationship between drug sensitivity and prognostic factors, which can be useful in the preclinical evaluation of new cytotoxic drugs. Furthermore, the results suggest that the pyrimidine analogues cytarabine and gemcitabine, as well as the anthracycline idarubicin, may have a role in the treatment of CLL. The new cyanoguanidine CHS 828 is highly active in CLL cells and appears to be non cross-resistant with standard drugs. The poorer prognosis in patients with CLL cells with unmutated IgV_H genes can not be explained by increased chemoresistance.</p>

Despite major advances in the understanding of the biology of chronic lymphocytic leukaemia (CLL), progress in improving its treatment has been limited and it still remains an incurable disorder. In the present research, we have performed in vitro drug sensitivity testing of primary CLL cells for preclinical evaluation of cytotoxic drugs, using the fluorometric microculture cytotoxicity assay (FMCA). The tumour type-specific activities of 14 standard drugs, evaluated in vitro on tumour cells from patients with CLL and acute leukaemias, were in good agreement with their known clinical activities. A correlation between drug treatment and development of cellular drug resistance was demonstrated in CLL, but not in the acute leukaemias. Moreover, the nucleoside analogues fludarabine, cladribine, cytarabine and gemcitabine, as well as the anthracycline idarubicin, were highly active in CLL cells. A new cytotoxic drug candidate, CHS 828, was evaluated in primary cell cultures from a broad spectrum of tumours. CHS 828 was highly active against haematological malignancies in vitro, especially CLL, but also against some solid tumours. The drug appeared to be non cross-resistant with standard drugs. In addition, the relationship between drug sensitivity in vitro and a recently described prognostic factor in CLL, the mutational status of the immunoglobulin variable heavy chain (IgVH) gene, was evaluated. Interestingly, cells with unmutated IgVH genes were more chemosensitive than the mutated cells. In summary, our results indicate that in vitro studies on tumour cellsfrom leukaemia patients may yield considerable information regarding the activity, mechanisms of action and cross-resistance of cytotoxic drugs, as well as concerning the relationship between drug sensitivity and prognostic factors, which can be useful in the preclinical evaluation of new cytotoxic drugs. Furthermore, the results suggest that the pyrimidine analogues cytarabine and gemcitabine, as well as the anthracycline idarubicin, may have a role in the treatment of CLL. The new cyanoguanidine CHS 828 is highly active in CLL cells and appears to be non cross-resistant with standard drugs. The poorer prognosis in patients with CLL cells with unmutated IgVH genes can not be explained by increased chemoresistance.