| 1. |
- Christensen, Kjeld, 1957-
(författare)
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Platelet Activation and Inhibition in Connection with Vascular Stents
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes the Chandler loop, which makes it possible to conduct studies in vitro of molecular and cellular interactions between whole blood and stents. It was possible to monitor activation and inhibition of the cascades systems, leukocytes and platelets by combining different platelet inhibitors and heparin coating of stents. The clinical study was performed on patients with ACS undergoing PCI and stent implantation. In this study platelet activation markers P-selectin, and αIIb/β3 as well as inflammatory markers were followed from baseline during the first 48 hours post-PCI. The same parameters were evaluated in healthy controls for comparison at baseline. In vitro: The activation of blood in the Chandler loops were more pronounced for unmodified stent grafts than for partially heparin coated stent grafts. Heparin coated stent grafts dreated the same activation as the loops alone. This indicated that the Chandler loop system was a feasible tool for evaluation of blood compability of stents. Heparin coating of stents significantly reduced TAT, CD11b and platelet activation. The combination of a heparin coated stent and abciximab reduced TAT and contact activation, as compared to abciximab or heparin coating alone. Heparin coating of stents in combination with AR-C69931MX resulted in a significant reduction in TAT and preservation of the platelet count but had no effect on contact activation. Clinical study: Abciximab resulted in an almost total inhibition of fibrinogen binding to platelets and persisted throughout the observation period. Clopidogrel effectscould be observed at four hours but was more pronounced at 24 hours. P-selectin expression did not differ over time between groups, indicating that platelet activation with α-granule secretion was not affected by abciximab treatment. The hs-CRP, C3a and sC5b-9 levels increased 24 to 48 hours after PCI in patients with ACS. FXIIa-C1inh was reduced in ACS patients receiving abciximab as compared to controls. The elevated bFGF levels at baseline returned to the levels observed in controls four hours after PCI and stent implantation, whereas an increase in VEGF was observed 24 hours post-PCI.
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| 2. |
- Eriksson, Anna, 1977-
(författare)
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Studies of New Signal Transduction Modulators in Acute Myeloid Leukemia
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Acute myeloid leukemia (AML) is a life-threatening malignant disorder with dismal prognosis. AML is characterized by frequent genetic changes involving tyrosine kinases, normally acting as important mediators in many basic cellular processes. Due to the overexpression and frequent mutations of the FMS-like receptor tyrosine kinase 3 (FLT3) in AML, this tyrosine kinase receptor has become one of the most sought after targets in AML drug development.In this thesis, we have used a combination of high-throughput screens, direct target interaction assays and sequential cellular screens, including primary patient samples, as an approach to discover new targeted therapies. Gefitinib, a previously known inhibitor of epidermal growth factor receptor and the two novel tyrosine kinase inhibitors AKN-032 and AKN-028, have been identified as compounds with cytotoxic activity in AML.AKN-028 is a potent inhibitor of FLT3 with an IC50 value of 6 nM in an enzyme assay, but also displaying in vitro activity in a variety of primary AML samples, irrespective of FLT3 mutation status or quantitative FLT3 expression. AKN-028 shows a sequence dependent in vitro synergy when combined with standard cytotoxic agents cytarabine or daunorubicin, with better efficacy when cells are exposed to standard chemotherapy simultaneously or for 24 hours prior to adding AKN-028. Antagonism is observed when cells are pre-treated with AKN-028, possibly explained by the cell cycle arrest induced by the compound. In vivo cytotoxic activity and good oral bioavailability have made AKN-028 a candidate drug for clinical studies and the compound is presently investigated in an international two-part multicenter phase I/II study.Results from microarray studies performed to further elucidate the mechanism of action of AKN-028, revealed significantly altered gene expression induced by AKN-028 in both AML cell lines and in primary AML cells, with an enrichment of the Myc pathway among the downregulated genes.Furthermore, tyrosine kinase activity profiling shows a dose-dependent kinase inhibition by AKN-028 in all AML samples tested. Interestingly, cells with a high overall kinase activity were more sensitive to AKN-028. Provided conformation in a larger set of samples, kinase activity profiling may give useful information in individualizing treatment of patients with AML.
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| 3. |
- Haglund, Caroline, 1981-
(författare)
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Integrating Efficacy and Toxicity in Preclinical Anticancer Drug Development : Methods and Applications
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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
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| 4. |
- Hovstadius, Peter, 1962-
(författare)
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Preclinical and Clinical Development of the Novel Cyanoguanidine CHS 828 for Cancer Treatment
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 5. |
- Karlsson, Henning, 1985-
(författare)
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New preclinical strategies for characterization and development of anticancer drugs
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Increased understanding of the molecular mechanisms underlying cancer development has shifted drug discovery towards target driven drug development the last decades, but the development of effective cancer drugs has been hampered by the lack of predictive preclinical models. 3-D cultures, considered to more accurately reflect solid tumors in vivo, have been proposed as one way to increase the predictability of clinical efficacy in cancer drug discovery and development.The aims of this thesis were to improve preclinical models for cancer drug development, with focus on colorectal cancer (CRC) and use of multicellular tumor spheroids (MCTS), and also to mechanistically characterize some potentially new anticancer drugs (papers I – IV). The most important technical improvement was the development of direct measurement of green fluorescent protein (GFP) marked cells in spheroids, simplifying live collection of viability data and enabling high-throughput screening (HTS) in the MCTS model (paper I). In paper III and IV, the 3-D model was adapted to enable studies on the interaction between drugs and radiation. Two potentially new anticancer drugs, VLX50 and VLX60, were mechanistically characterized. VLX60, a novel copper containing thiosemicarbazone, induced reactive oxygen species (ROS) formation, was selectively active against BRAF mutated colon cancer cells and exhibited anticancer activity in vivo (paper II). Furthermore, two potentially new anticancer drugs were found suitable for further development for use in combination with radiation (papers III and IV). In paper III, synergy with radiation in spheroids compared to monolayer cultured colon cancer cells was shown with the novel iron-chelating inhibitor of oxidative phosphorylation, VLX600. In paper IV, the antiprotozoal drug nitazoxanide was shown to sensitize quiescent clonogenic colon cancer cells to radiation.In conclusion, introduction of measurement of fluorescence of GFP marked cells in spheroids makes clinically relevant 3-D models feasible for HTS experiments and characterization of candidate drugs and radiosensitizers in early cancer drug discovery and development. VLX60 has several characteristics suitable for further development into a cancer drug, notably against BRAF mutated colorectal cancer cells. VLX600 and nitazoxanide show radiosensitizing properties making them promising for further development for use as cancer drugs in combination with radiation.
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| 6. |
- Kashif, Muhammad
(författare)
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Integrated Computational and Experimental Approaches for Accelerated Drug Combination Discovery and Development : Applications in Cancer Pharmacology
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Today the norm in modern cancer treatment is to use different forms of drug combinations. Recently anti-cancer treatment using drug combinations has gained increased attention due to the outstanding pharmacotherapeutic opportunities provided by combination therapies. However, the potential of this field is largely unexplored, partly due to the complexities associated with the astronomical number of possible combinations and partly due to the lack of means for quantifying clinically relevant adverse side effects in the early stages of the combination discovery and development process. This has resulted in relatively limited progress in this area. Motivated by this unfortunate state-of-affairs, the research reported in this thesis was aimed at developing and implementing computational and experimental methods to facilitate and accelerate the discovery and development of anti-cancer therapies. In paper I, the largely overlooked concept of therapeutic synergy is re-introduced and demonstrated to be useful already at the level of combination discovery by taking both curative and adverse effects into account. In paper II, a semiautomatic combination discovery platform was developed based on a tailored programming of a pipetting robot system and application of a new in-house developed combination search algorithm, the therapeutic algorithmic combinatorial screen (TACS) algorithm. TACS seems to be the first algorithm of its kind that takes experimental variability into account during the iterative search process. The semiautomatic hardware platform along with TACS can perform de novo or knowledge based combination drug discovery and development without brute force comprehensive search efforts. One promising discovery made using this platform is a combination of the drugs 17-AAG, afungin and trichostatin a for treatment of colorectal cancer carcinoma (CRC). In paper III, an algorithm is developed and applied in order to use single drug induced systemic gene expression profiles for rational drug combination design by assuming additive combination effects. The resulting algorithm, combo-CMap, is applied and validated using a slightly extended version of the freely available Connectivity Map (CMap) database which is currently containing 6190 chemically induced mRNA gene expression signatures. In paper IV, a software (R package) was developed and applied to perform improved synergy/antagonism analysis, in particular joint Loewe and Bliss analyses while taking associated experimental variability into account using non-parametric statistics including bootstrap intervals. Applying this software to the synergy analysis of interaction effects among clinically used and/or relevant drugs in CRC cell lines revealed complex patterns of synergy and antagonism. In conclusion, the work presented here offers important contributions and findings that may accelerate and/or improve different parts of the field of drug combination discovery and development.
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| 7. |
- Palle, Josefine, 1964-
(författare)
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Optimizing Chemotherapy in Childhood Acute Myeloid Leukemia
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Despite major advances in our understanding of the biology of childhood acute myeloid leukemia (AML) and the development of new cytotoxic drugs, the prognosis of long-term survival is still only 60-65 %.In the present research, we studied the pharmacokinetics of drugs used in the induction therapy of childhood AML and performed in vitro drug sensitivity testing of leukemic cells from children with AML.The aims of the studies were to correlate the results of the analysis to biological and clinical parameters and to identify subgroups of AML with specific drug sensitivity profiles in order to better understand why treatment fails in some patients and how therapy may be improved.Blood samples were analysed to study the pharmacokinetics of doxorubicin (n=41), etoposide (n=45) and 6-thioguanine (n=50). Doxorubicin plasma concentration and total body clearance were correlated to the effect of induction therapy, and doxorubicin plasma concentration was an independent factor for complete remission, both in univariate and multivariate analysis including sex, age, and white blood cell count at diagnosis. For etoposide and 6-thioguanine no correlation was found between pharmacokinetics and clinical effect. Children with Down syndrome (DS) tended to reach higher blood concentrations of etoposide and thioguanine nucleotides, indicating that dose reduction may be reasonable to reach the same drug exposure as in children without DS.Leukemic cells from 201 children with newly diagnosed AML, 15 of whom had DS, were successfully analysed for in vitro drug sensitivity by the fluorometric microculture cytotoxicity assay (FMCA). We found that samples from children with DS were highly sensitive to most drugs used in AML treatment. In non-DS children, the t(9;11) samples were significantly more sensitive to cytarabine (p=0.03) and doxorubicin (p=0.035) than other samples. The findings might explain the very favorable outcome reported in children with DS and t(9;11)-positive AML. A specific drug resistance profile was found for several other genetic subgroups as well. A detailed study of MLL-rearranged leukemia showed that cellular drug sensitivity is correlated both to partner genes and cell lineage, findings that support the strategy of contemporary protocols to include high-dose cytarabine in the treatment of patients with MLL-rearrangement, both in AML and acute lymphoblastic leukemia (ALL).Our results indicate that drug resistance and pharmacokinetic studies may yield important information regarding drug response in different sub-groups of childhood AML, helping us to optimize future chemotherapy in childhood AML.
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| 8. |
- Rickardson, Linda, 1980-
(författare)
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New Methods to Screen for Cancer Drugs and to Evaluate their Mechanism of Action
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cancer is a common disease and due to problems with resistance against cancer drugs and the limited benefit from chemotherapy in many diagnoses, there is a need to develop new cancer drugs. In this thesis new methods to screen for cancer drugs and to evaluate their mechanism of action are discussed. In Paper I, it was found that by studying the gene expression of a cell line panel and combining the data with sensitivity data of a number of cytotoxic drugs, it was possible to cluster compounds according to mechanism of action as well as identifying genes associated with chemosensitivity. In Paper II, studies of compounds with selective activity in drug-resistant cell lines revealed the glucocorticoids as a group of interesting compounds. The glucocorticoid receptor was overexpressed in 8226/Dox40 and the difference in sensitivity was abolished when the cells were treated with a glucocorticoid receptor antagonist. In Paper III, an image-based screening method for new proteasome inhibitors was successfully developed and the compounds disulfiram, PDTC and NSC 95397 were identified as inhibitors of the proteasome. In Paper IV, disulfiram and PDTC were shown to induce cytotoxic activity, to inhibit the activation of the transcription factor NFkappaB and to inhibit the degradation of proteins normally degraded by the proteasome. In Paper V, NSC 95397 was shown to be cytotoxic to all cells in the resistance-based cell line panel as well as to patient samples from a variety of cancer diagnoses. Connectivity Map was successfully used as a tool to propose a new mechanism of action of NSC 95397. The gene expression induced by NSC 95397-treatment was similar to that induced by several proteasome inhibitors not present in the Connectivity Map.
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| 9. |
- Selvin, Tove
(författare)
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Preclinical tumor-immune modeling : For the identification of immunomodulatory drugs
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- For a long time, the field of cancer research was dominated by a tumor cell-centric view. That, however, changed once it became recognized that medical cancer treatment is largely influenced by the combined effect exerted on both cancer and immune cells. In this work, we aimed to develop and apply preclinical model systems for the identification and evaluation of immunomodulatory anti-cancer agents. In Paper I, we employed single-cell RNA sequencing (scRNA-seq) to investigate immunological effects of trifluridine (FTD), a nucleoside analogue used for the treatment of colorectal cancer (CRC). The study revealed that while FTD induces immunogenic cell death (ICD), it may also attenuate T cell-mediated antitumor responses. In paper II and III, we developed and applied a phenotypic screening platform based on a miniaturized tumor-immune model. In paper II, aiming to identify immunological effects of clinical relevance and provide a reference point for screening novel compound libraries, the model system was used to assess a broad panel of standard anticancer agents. In paper III, the platform was used to screen a drug library containing 1280 small molecule drugs, all approved by the FDA or other agencies. Using this approach, statins were identified as enhancers of immune cell-mediated cancer cell killing. Finally, in paper IV, we developed the immuno-oncology hollow fiber assay (HFA) with the goal of bridging the gap between cell based in vitro assays and more complex mouse models for evaluation of immuno-oncological agents. The HFA is an in vivo assay in which semipermeable fibers are filled with cancer cells and implanted in rodents. We further developed the HFA to incorporate both cancer and immune cells. This novel assay demonstrated the potential to capture immune-mediated cancer cell killing in vivo within a matter of days. Collectively, this work provides a research approach for immuno-oncology drug screening, in vitro validation, and initial in vivo evaluation.
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| 10. |
- Senkowski, Wojciech
(författare)
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High-throughput screening using multicellular tumor spheroids to reveal and exploit tumor-specific vulnerabilities
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- High-throughput drug screening (HTS) in live cells is often a vital part of the preclinical anticancer drug discovery process. So far, two-dimensional (2D) monolayer cell cultures have been the most prevalent model in HTS endeavors. However, 2D cell cultures often fail to recapitulate the complex microenvironments of in vivo tumors. Monolayer cultures are highly proliferative and generally do not contain quiescent cells, thought to be one of the main reasons for the anticancer therapy failure in clinic. Thus, there is a need for in vitro cellular models that would increase predictive value of preclinical research results. The utilization of more complex three-dimensional (3D) cell cultures, such as multicellular tumor spheroids (MCTS), which contain both proliferating and quiescent cells, has therefore been proposed. However, difficult handling and high costs still pose significant hurdles for application of MCTS for HTS.In this work, we aimed to develop novel assays to apply MCTS for HTS and drug evaluation. We also set out to identify cellular processes that could be targeted to selectively eradicate quiescent cancer cells. In Paper I, we developed a novel MCTS-based HTS assay and found that nutrient-deprived and hypoxic cancer cells are selectively vulnerable to treatment with inhibitors of mitochondrial oxidative phosphorylation (OXPHOS). We also identified nitazoxanide, an FDA-approved anthelmintic agent, to act as an OXPHOS inhibitor and to potentiate the effects of standard chemotherapy in vivo. Subsequently, in Paper II we applied the high-throughput gene-expression profiling method for MCTS-based drug screening. This led to discovery that quiescent cells up-regulate the mevalonate pathway upon OXPHOS inhibition and that the combination of OXPHOS inhibitors and mevalonate pathway inhibitors (statins) results in synergistic toxicity in this cell population. In Paper III, we developed a novel spheroid-based drug combination-screening platform and identified a set of molecules that synergize with nitazoxanide to eradicate quiescent cancer cells. Finally, in Paper IV, we applied our MCTS-based methods to evaluate the effects of phosphodiesterase (PDE) inhibitors in PDE3A-expressing cell lines.In summary, this work illustrates how MCTS-based HTS yields potential to reveal and exploit previously unrecognized tumor-specific vulnerabilities. It also underscores the importance of cell culture conditions in preclinical drug discovery endeavors.
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