<p>The antitumoural cyanoguanidine CHS 828 has shown promising activity in a number of preclinical and clinical studies. However, the mechanisms underlying the cell death induced by CHS 828 has not been clarified. This thesis describes in vitro studies of the cellular pharmacology of CHS 828.</p><p>CHS 828 induced cell death with necrosis like features in the lymphoma cell line U-937 GTB. Addition of 3-aminobenzamide, an inhibitor of ADP-ribosylation, resulted in a decreased sensitivity to CHS 828 and a shift in the mode of cell death towards apoptosis. </p><p>Mouse fibroblasts lacking the enzyme PARP-1 were more sensitive to CHS 828 compared to normal fibroblasts. CHS 828 was able to induce p53 in normal fibroblasts but this effect does not seem to be necessary to induce cell death.</p><p>Characterization of two CHS 828 resistant cell lines indicated that they were selectively resistant to cyanoguanidines. Known mechanisms of anticancer drug resistance did not seem to account for the cyanoguanidine resistance. One possible resistance mediating protein, which was upregulated in the resistant cells, was epidermal fatty acid binding protein.</p><p>A novel high content screening assay was also developed. The assay was shown to be suitable both for screening of potential novel antitumoural substances as well for mechanistic studies. In the assay, CHS 828 induced caspase-3 activity and reduction in mitochondrial membrane potential, both signs of apoptosis, in U-937 GTB cells. However, nuclei in exposed cells did not show nuclear fragmentation, one of the hallmarks of apoptosis.</p><p>CHS 828 was also shown to indirectly inhibit the proteasome activity in U-937 GTB cells. </p><p>In conclusion, the results presented provide new insights into the metabolic and molecular events involved in cell death induced by CHS 828.</p>

<p>CHS 828 is a cyanoguanidine with anti-tumour properties which has shown promising effects in several preclinical models. This thesis describes both preclinical and clinical studies aiming to investigate disease specific activity, clinical tolerability and efficacy of CHS 828.</p><p>In paper I we investigated CHS 828 activity in a cell line panel with human myeloma cells, three of these cell-lines were also tested in vivo using a hollow fibre rat-model. In paper II we investigated CHS 828 activity in primary human tumour samples from patients. CHS 828 showed an effect on all tumour cell types tested both the primary human tumour samples and the myeloma cell lines. Notably, CHS 828 showed a high relative in vitro activity against tumour cells from chronic lymphocytic leukaemia and high-grade lymphoma. </p><p>In a phase I trial we determined the maximum tolerated dose (MTD) of CHS 828. Haematological toxicity was generally mild and dominated by transient thrombocytopenia and lymphocytopenia. Non-haematological toxicity was mostly of gastrointestinal origin. The recommended phase two dose (RPTD) of CHS 828 was estimated to be 20 mg once daily for five days in cycles of 28 days duration.</p><p>In a phase II trial we investigated the effect of CHS 828 on patients diagnosed with B-CLL. In total 12 patients were enrolled. CHS 828 was found to be well tolerated and the most common haematological toxicity was thrombocytopenia. Non-haematological toxicities were generally mild. Transient decreases in lymphocyte counts could be discerned coinciding with drug dosing, but no sustained clinical responses could be achieved.</p><p>In conclusion, CHS 828 demonstrated marked effects in the preclinical investigations suggesting haematological malignancies as the main target. The clinical phase I study established a safe dose and the subsequent phase II trial in B-CLL patients showed biological effect but with no clinical disease response. </p>

The antitumoural cyanoguanidine CHS 828 has shown promising activity in a number of preclinical and clinical studies. However, the mechanisms underlying the cell death induced by CHS 828 has not been clarified. This thesis describes in vitro studies of the cellular pharmacology of CHS 828. CHS 828 induced cell death with necrosis like features in the lymphoma cell line U-937 GTB. Addition of 3-aminobenzamide, an inhibitor of ADP-ribosylation, resulted in a decreased sensitivity to CHS 828 and a shift in the mode of cell death towards apoptosis. Mouse fibroblasts lacking the enzyme PARP-1 were more sensitive to CHS 828 compared to normal fibroblasts. CHS 828 was able to induce p53 in normal fibroblasts but this effect does not seem to be necessary to induce cell death. Characterization of two CHS 828 resistant cell lines indicated that they were selectively resistant to cyanoguanidines. Known mechanisms of anticancer drug resistance did not seem to account for the cyanoguanidine resistance. One possible resistance mediating protein, which was upregulated in the resistant cells, was epidermal fatty acid binding protein. A novel high content screening assay was also developed. The assay was shown to be suitable both for screening of potential novel antitumoural substances as well for mechanistic studies. In the assay, CHS 828 induced caspase-3 activity and reduction in mitochondrial membrane potential, both signs of apoptosis, in U-937 GTB cells. However, nuclei in exposed cells did not show nuclear fragmentation, one of the hallmarks of apoptosis. CHS 828 was also shown to indirectly inhibit the proteasome activity in U-937 GTB cells. In conclusion, the results presented provide new insights into the metabolic and molecular events involved in cell death induced by CHS 828.

CHS 828 is a cyanoguanidine with anti-tumour properties which has shown promising effects in several preclinical models. This thesis describes both preclinical and clinical studies aiming to investigate disease specific activity, clinical tolerability and efficacy of CHS 828. In paper I we investigated CHS 828 activity in a cell line panel with human myeloma cells, three of these cell-lines were also tested in vivo using a hollow fibre rat-model. In paper II we investigated CHS 828 activity in primary human tumour samples from patients. CHS 828 showed an effect on all tumour cell types tested both the primary human tumour samples and the myeloma cell lines. Notably, CHS 828 showed a high relative in vitro activity against tumour cells from chronic lymphocytic leukaemia and high-grade lymphoma. In a phase I trial we determined the maximum tolerated dose (MTD) of CHS 828. Haematological toxicity was generally mild and dominated by transient thrombocytopenia and lymphocytopenia. Non-haematological toxicity was mostly of gastrointestinal origin. The recommended phase two dose (RPTD) of CHS 828 was estimated to be 20 mg once daily for five days in cycles of 28 days duration. In a phase II trial we investigated the effect of CHS 828 on patients diagnosed with B-CLL. In total 12 patients were enrolled. CHS 828 was found to be well tolerated and the most common haematological toxicity was thrombocytopenia. Non-haematological toxicities were generally mild. Transient decreases in lymphocyte counts could be discerned coinciding with drug dosing, but no sustained clinical responses could be achieved. In conclusion, CHS 828 demonstrated marked effects in the preclinical investigations suggesting haematological malignancies as the main target. The clinical phase I study established a safe dose and the subsequent phase II trial in B-CLL patients showed biological effect but with no clinical disease response.