Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 5 uppsatser.

<p>Antitumour properties of the cyanoguanidine CHS 828 and analogues were discovered in 1997. CHS 828 is presently in clinical phase I/II trials. This thesis encompasses in vitro studies of the kinetics and mode of cell death induced in the human cell line U-937 GTB, by CHS 828 and the standard antitumour drug etoposide.</p><p>Etoposide induces apoptosis in U-937 GTB within 4 h. The cells exhibited apoptotic morphology, including condensed and fragmented nuclei and formation of apoptotic bodies, activation of caspase 3 and 8, and DNA fragmentation, visualised by TdT-mediated dUTP nick end-labelling (TUNEL).</p><p>CHS 828 induced few and weak signs of apoptosis. Metabolic activity was the only parameter affected during the first 24 h of exposure. After ~30 h, proliferation (DNA synthesis) and protein synthesis ceased, and viability started to decrease towards 10% at 72 h. Morphology and ultrastructure of dying/dead cells showed predominant necrosis. The decrease in viability was postponed by protein synthesis inhibition or maintenance of ATP levels by 3-aminobenzamide. In addition, 3-aminobenzamide switched morphology towards apoptosis. </p><p>Continuous co-exposure to CHS 828 and etoposide resulted in impressive cell kill synergy in U-937 GTB cells at effect levels of 30-70%. Pre-exposure to CHS 828 for 18 h or more, on the other hand, resulted in diminished cell kill and inability to activate the apoptotic machinery upon etoposide stimulation, evaluated by morphology and caspase activity.</p><p>In summary, CHS 828 induced cell death is predominantly non-apoptotic, does not involve caspases and can be postponed by maintained protein synthesis and ATP levels.</p>

<p>Neuroblastoma is one of the most common solid cancers of early childhood. In Sweden, approximately 10-15 cases occur annually. The overall five-year neuroblastoma survival in Europe is approximately 45%. Since cancer treatment involves drugs with risks of side effects in the growing child, there is a need for more effective and less toxic drugs. One new approach in cancer treatment is inhibition of tumor angiogenesis, i.e., of new blood vessel growth into the tumor. An angiogenesis inhibitor may be combined with cytostatic drugs to enhance the efficacy. The aim of this study was to investigate how drugs could be used to inhibit angiogenesis and tumor growth in a xenograft model of human neuroblastoma in nude mice. </p><p>The tumors express the angiogenesis stimulator vascular endothelial growth factor (VEGF) on both protein and mRNA levels. The angiogenesis inhibitors SU5416 (an inhibitor of VEGF signalling) and TNP-470 (an inhibitor of endothelial cell proliferation) inhibited angiogenesis in our model. TNP-470, however, inhibited angiogenesis without significant reduction of the tumor growth, in contrast to SU5416. </p><p>We also discovered that the cytostatic drug CHS 828 could cause regression of neuroblastoma tumors in the model when given orally at a low daily dose, alone or in combination with the angiogenesis inhibitor SU5416 or TNP-470. </p><p>Furthermore, a new use of the cardiac glycoside digoxin was found. Digoxin inhibited FGF-2 -stimulated bovine capillary endothelial cell growth in vitro, and inhibited angiogenesis in vivo in the chick chorioallantoic membrane assay (CAM). It also inhibited neuroblastoma growth by approximately 50% in our neuroblastoma model. </p><p>In conclusion, CHS 828 and digoxin represent two classes of drugs with potent antitumor effects that may be valuable in treatment of neuroblastoma, either alone or in combination with angiogenesis inhibitors.</p>

<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>The novel cyanoguanidine CHS 828 has shown promising antitumor activity in many in vitro and in vivo studies. </p><p>The long-term 14 days in vitro hollow fiber cultures, where tumor cells from different tumor cell lines were cultured inside semipermeable fibers, were more resistant to CHS 828 and other cytotoxic drugs than the shorter-term 3 days cultures. CHS 828 was generally more effective against haematological than solid tumor cells from both cell lines and patients samples. </p><p>In vivo, the hollow fibers were implanted into immunocompetent rats and the pharmacokinetics, tumor response and/or toxicity (pharmacodynamics) of CHS 828 were successfully assayed. CHS 828 showed higher activity in this model when a more protracted schedule was used. The quantitative relationships between dose, plasma concentration and response (PK/PD model) developed for CHS 828 explained this phenomenon partly by dose-dependent fraction absorbed and partly by a schedule-dependent pharmacodynamic effect.</p><p>Modelling of the in vitro CHS 828 and standard cytotoxic drugs concentration-time effect data in different tumor cell types and characterization of pattern of change of the potency and the slope of the concentration-time effect curves were performed. The results suggest two different mechanisms of action for CHS 828 and that CHS 828 cytotoxicity may depend on the schedule used.</p><p>The NF-kB pathway that regulates the transcription of anti-apoptotic genes proved to be inhibited by CHS 828 in different tumor cell lines and the inhibition was correlated to the cell death induced by this agent. CHS 828 did not seem to induce the NF-kB inhibition by affecting the proteasome activity. </p><p>The in vitro and in vivo hollow fiber methods were also used successfully to evaluate the new paclitaxel formulation, Pacliex. Pacliex had a similar activity to that of the clinically used formulation Taxol®.</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>

Antitumour properties of the cyanoguanidine CHS 828 and analogues were discovered in 1997. CHS 828 is presently in clinical phase I/II trials. This thesis encompasses in vitro studies of the kinetics and mode of cell death induced in the human cell line U-937 GTB, by CHS 828 and the standard antitumour drug etoposide. Etoposide induces apoptosis in U-937 GTB within 4 h. The cells exhibited apoptotic morphology, including condensed and fragmented nuclei and formation of apoptotic bodies, activation of caspase 3 and 8, and DNA fragmentation, visualised by TdT-mediated dUTP nick end-labelling (TUNEL). CHS 828 induced few and weak signs of apoptosis. Metabolic activity was the only parameter affected during the first 24 h of exposure. After ~30 h, proliferation (DNA synthesis) and protein synthesis ceased, and viability started to decrease towards 10% at 72 h. Morphology and ultrastructure of dying/dead cells showed predominant necrosis. The decrease in viability was postponed by protein synthesis inhibition or maintenance of ATP levels by 3-aminobenzamide. In addition, 3-aminobenzamide switched morphology towards apoptosis. Continuous co-exposure to CHS 828 and etoposide resulted in impressive cell kill synergy in U-937 GTB cells at effect levels of 30-70%. Pre-exposure to CHS 828 for 18 h or more, on the other hand, resulted in diminished cell kill and inability to activate the apoptotic machinery upon etoposide stimulation, evaluated by morphology and caspase activity. In summary, CHS 828 induced cell death is predominantly non-apoptotic, does not involve caspases and can be postponed by maintained protein synthesis and ATP levels.

Neuroblastoma is one of the most common solid cancers of early childhood. In Sweden, approximately 10-15 cases occur annually. The overall five-year neuroblastoma survival in Europe is approximately 45%. Since cancer treatment involves drugs with risks of side effects in the growing child, there is a need for more effective and less toxic drugs. One new approach in cancer treatment is inhibition of tumor angiogenesis, i.e., of new blood vessel growth into the tumor. An angiogenesis inhibitor may be combined with cytostatic drugs to enhance the efficacy. The aim of this study was to investigate how drugs could be used to inhibit angiogenesis and tumor growth in a xenograft model of human neuroblastoma in nude mice. The tumors express the angiogenesis stimulator vascular endothelial growth factor (VEGF) on both protein and mRNA levels. The angiogenesis inhibitors SU5416 (an inhibitor of VEGF signalling) and TNP-470 (an inhibitor of endothelial cell proliferation) inhibited angiogenesis in our model. TNP-470, however, inhibited angiogenesis without significant reduction of the tumor growth, in contrast to SU5416. We also discovered that the cytostatic drug CHS 828 could cause regression of neuroblastoma tumors in the model when given orally at a low daily dose, alone or in combination with the angiogenesis inhibitor SU5416 or TNP-470. Furthermore, a new use of the cardiac glycoside digoxin was found. Digoxin inhibited FGF-2 -stimulated bovine capillary endothelial cell growth in vitro, and inhibited angiogenesis in vivo in the chick chorioallantoic membrane assay (CAM). It also inhibited neuroblastoma growth by approximately 50% in our neuroblastoma model. In conclusion, CHS 828 and digoxin represent two classes of drugs with potent antitumor effects that may be valuable in treatment of neuroblastoma, either alone or in combination with angiogenesis inhibitors.

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.

The novel cyanoguanidine CHS 828 has shown promising antitumor activity in many in vitro and in vivo studies. The long-term 14 days in vitro hollow fiber cultures, where tumor cells from different tumor cell lines were cultured inside semipermeable fibers, were more resistant to CHS 828 and other cytotoxic drugs than the shorter-term 3 days cultures. CHS 828 was generally more effective against haematological than solid tumor cells from both cell lines and patients samples. In vivo, the hollow fibers were implanted into immunocompetent rats and the pharmacokinetics, tumor response and/or toxicity (pharmacodynamics) of CHS 828 were successfully assayed. CHS 828 showed higher activity in this model when a more protracted schedule was used. The quantitative relationships between dose, plasma concentration and response (PK/PD model) developed for CHS 828 explained this phenomenon partly by dose-dependent fraction absorbed and partly by a schedule-dependent pharmacodynamic effect. Modelling of the in vitro CHS 828 and standard cytotoxic drugs concentration-time effect data in different tumor cell types and characterization of pattern of change of the potency and the slope of the concentration-time effect curves were performed. The results suggest two different mechanisms of action for CHS 828 and that CHS 828 cytotoxicity may depend on the schedule used. The NF-kB pathway that regulates the transcription of anti-apoptotic genes proved to be inhibited by CHS 828 in different tumor cell lines and the inhibition was correlated to the cell death induced by this agent. CHS 828 did not seem to induce the NF-kB inhibition by affecting the proteasome activity. The in vitro and in vivo hollow fiber methods were also used successfully to evaluate the new paclitaxel formulation, Pacliex. Pacliex had a similar activity to that of the clinically used formulation Taxol®.