Pharmaceutical Biotechnology: knowledge for health
Head: Prof. Janko Kos
The research work of the programme group is part of the advanced trends in science aimed at increasing our understanding of living processes, improving health care and creating a sustainable environment. Understanding the basic mechanisms of cell function will also help to reveal mechanisms of the development and progression of diseases. We are aware that understanding the main factors in these mechanisms and processes enables the identification of new targets for more effective diagnosis and therapy. Recent achievements of the members of the programme group, reflected in numerous publications, citations and patents, constitute a significant contribution to universal understanding. An important part of the work is screening and developing new potential drugs and new diagnostic, analytical and biotechnological procedures. Moreover, our group is introducing into our research area the most recent techniques and methods. The collaboration with other researchers in Slovenia and worldwide guarantees rapid transfer of knowledge, good science and valuable applications.
Genes, hormones and mental changes in metabolic disorders
Head: Prof. Janez Preželj
The programme is based on the results of our previous research in the field of osteoporosis, diabetes and polycystic ovary syndrome (PCO).
In osteoporosis research the approach will include determinations of local regulators of bone metabolism (osteoprotegerin, RANK, RANKL, interleukine 1, TNF, kathepsin K). Expression of genes relevant to bone metabolism will be measured in bone tissue provided during hip surgery in osteoporotic patients. We hope to provide new insight into pathophysiology of osteoporosis at cellular level. We will proceed with our research in genetics of osteoporosis to get possible predictors of therapy response and of risk for osteoporosis.
We will proceed investigation on insulin resistance in AIDS and PCO, trying to get information of possible differences compared to patients with diabetes mellitus type 2. In patients with PCO we plan to follow changes in insulin resistance after therapy with metpformin or rosiglitazone.
In patients with diabetes mellitus our research will focus on endothelin, endothelin gene polymorphism and adiponectin and their role in pathogenesis of diabetic nephropathy.
The other aspect of diabetes research will include mental disorders in this population. We will screen outpatient population by means of CES-D and SCL90. These tests were not yet used in diabetes mellitus patients.
Systemic autoimmune diseases
Head: Assist. prof. Snežna Sodin Šemrl
The field of immunology is one of the most rapidly growing areas of scientific experimental and clinical research. Foundations of immunology lay in molecular biology, biochemistry, biophysical chemistry, molecular biotechnology, molecular genetics and clinical medicine. In many countries, including Slovenia, rheumatology covers systemic autoimmune diseases. We have been involved in these studies for more than 15 years. Our recent program deals with three diseases in which the disturbances in humoral immunity play at least a very important if not a crucial role in the pathogenesis of the diseases. The program integrates
a/clinical and laboratory research of antiphospholipid syndrome and antiphospholipid antibodies,
b/clinical studies of Sjoegren's syndrome with special emphasise on salivary glands together with the detection of antibodies against sinthetic peptides of muscarinic acethylcholine receptors,
c/the epidemiological study of arterial hypertension in systemic sclerosis with the studies of functional characteristics of small and middle size artery walls.
Pharmaceutical Chemistry: planning, synthesis and valuation of active substances
Head: Prof. Danijel Kikelj
Importance of results of the research programme for the development of science also in 2010 remained in (i) discovery of novel drugs in important therapeutic areas and in (ii) development of methods for their design, synthesis and study of their molecular mechanism of action. The programme is expected to give an important contribution to discovery of innovative antibacterial drugs with novel mechanisms of action. Discovery of novel antibacterial drugs targeting intracellular steps of peptidoglycane biosynthesis is a great challenge to science due to a threatening bacterial resistance to currently used antibacterial drugs. There is an urgent need for development of new antithrombotic drugs due to high incidence of thrombotic diseases in developed countries. The concept of dual antithrombotic drugs, targeting thrombin or factor Xa and fibrinogen receptor which is being developed as a part of the current programme could lead to antithrombotic drugs of the future. Multiple modulators of integrin receptors possess a great potential as future antitumour and antithrombotic drugs as well. Intensive biochemical and immunological research of DC-SIGN receptor over the past few years provides an excellent base for the development of novel DC-SIGN inhibitors. With innovative glycomimetic approach we plan to perform some pioneer steps in the field of DC-SIGN inhibitors and antimicrobial therapy. Application of state-of-the-art technologies, interdisciplinary approach and strong international networking will enable a successful search for novel modulators of apoptosis and new targets that will help to understand apoptosis regulating mechanisms. New molecular tools for EPR spectroscopy, fluorescence spectroscopy and fluorescence microscopy are important for studies of mechanism of action of a number of drugs (e.g. inducing of apoptosis, influencing membrane domain composition, transmembrane signalling).
The importance of the proposed research programme for science is thus in (i) discovery of novel enzyme inhibitors and modulators of receptors as novel antibacterial, antithrombotic and antitumour agents with potential for further development in pharmaceutical industry, (ii) introduction of a paradigm of designed multiple ligands in the discovery of antibacterial, antithrombotic and antitumour drugs, (iii) identification of new target macromolecules involved in regulation of apoptosis and discovery of new apoptosis modulating compounds, (iv) new approaches to the design of multiple ligands, (v) new approaches to rational drug design and drug synthesis, (vi) development of new synthetic methods, (vii) development of new mimetics of peptides, sugars and lipids and (viii) development of new molecular tools for study of biological processes and biological macromolecules
ONGOING ARRS PROJECTS
Pharmacogenetic approaches to research, diagnosis and therapy of leukemia
Head: Prof. Irena Mlinarič Raščan
The proposed project is motivated by the requirement of translation of genomic, transcriptomic and metabolomic methodologies to clinical medicine with the goal to improve both diagnosis and treatment of pediatric leukemia patients.
State-of-the art and Objectives
Despite many advances in the understanding of cancer biology, therapeutic treatment of tumors remains essentially empiric. The main problem of chemotherapy is the narrow therapeutic index of antitumor agents that results in severe toxicity and low treatment outcome. The use of pharmacogenomics to individualize drug therapy offers the potential to improve drug effectiveness, reduce adverse side effects, and provide cost-effective pharmaceutical care.
Thiopurine drugs are used in the treatment of acute lymphoblast leukaemia (ALL), the most common childhood cancer. Our previous studies have delineated the genotype-to-phenotype correlation for the thiopurine S-methyltransferase (TPMT), which catalyzes methylation and thus deactivation of 6-MP, and further linked the low enzyme activity to the onset of severe toxicity of 6-MP. Beside genotype other factors also influence the activity of TPMT. Identification and understanding of factors influencing TPMT activity is crucial for the improvement of the efficacy and safety of ALL treatment. We therefore intend to further investigate the competitive metabolic routes for thiopurines. By dddressing the molecular mechanisms of thiopurine action and selected enzyme function we intend to search for a novel potential targets for new-generation of therapeutic agents.
Aims of the project
1.Identification of biological markers and their implementation in clinical practice
We will try to identify additional factors influencing TPMT activity., by testing the correlation between levels of methionine cytotoxic metabolites of 6-MP and TPMT activity.
2. Identification of novel therapeutically relevant biological targets
Delineation of the impact of TPMT on intracellular metabolism will allow the characterization of relevant enzymes, metabolites and differentially expressed genes as candidates for clinically relevant targets with potential roles in the modulation of cell- and tissue-specific processes (oxidative stress, methionine metabolism, DNA methylation).
3. Elucidation of molecular mechanism of thiopurine therapy response
The differential genome, transcriptome and metabolome analysis will be preformed on thiopurines resistant and non-resistant cells. The selected candidate genes, metabolites and enzymes will be implemented in clinical practice as diagnostic test for optimization of thiopurine therapy in ALL patients.
4. Development of 6-MP dosing algorithm. The dosing algorithm will enable the calculation of the optimal individual dose for each patient, according to his or her demographic, phenotypic and genotypic characteristics.
Relevance of the proposed project
The project will be conducted by an international, interdisciplinary team including partners from University of Ljubljana, Faculty of Pharmacy, Department of Pediatrics, University Clinical Center Ljubljana (dr. Janez Jazbec), Pediatric hospital St. Anna Kinderspital in Vienna, Austria (dr. Leo Kager), Estonian Genome Project and Institute for molecular and cell biology, University of Tartu (dr. Andres Metspalu), and Sackler Faculty of Medicine, University of Tel Aviv (dr. David Gurwitz). Translation of genomic, transcriptomic and metabolomic methodologies in the field of pharmacogenetics shall improve both diagnosis and treatment of pediatric leukemia patients. All of this indicates a high scientific and socio-economic relevance. Proposed approach and expertise of the group shall assure high-scientific relevance and accomplishments of project’s ambitious goals.
The influence of active transport and patients' genotypes on the therapeutic outcome of chronic myeloic leukemia
Head: Prof. Albin Kristl
Chronic myeloid leukaemia (CML) is a malignant neoplasm of hematopoietic stem cell and is classified among mieloproliferative diseases. 15-20% of adult patients with leukaemia have CML. The natural course of the disease lasts 3 – 5 years and has three phases: chronic, in which 85% of patients are diagnosed, followed by an accelerated period, leading to blast transformation. Since 2001, an era of molecular therapy of CML began with imatinib, the first tyrosine kinase inhibitor (TKI) on the market. Since then the eight-year survival of patients with CML is as high as 93% if only deaths related to CML are considered. Severe intolerance of imatinib occurs in less than 5% of patients while the primary resistance to treatment occurs in 25% of patients treated with imatinib. Several mechanisms of resistance exist and are intensely investigated. It seems that the resistance is either directly or indirectly (through increased emergence of TKI resistant clones) linked to insufficient drug concentration at the site of action. Such low concentrations, emergence of resistance and therapy failure were already linked to poor uptake of imatinib in the target cells by OCT1 transporters. The diffusion of imatinib from the gastrointestinal lumen to blood plasma, as well as its diffusion from blood plasma to target cells, are most likely highly dependent on transport protein activity.
In the proposed project we wish to explore the processes that determine imatinib concentrations at the site of action – that is in the target cells (mononuclear leukocytes). Our main focus will be on the molecular mechanisms of drug absorption from the gastrointestinal tract into the bloodstream and, regarding the clinical significance, its distribution between the blood plasma and the cytosol of target cells in the peripheral blood and in bone marrow. Therefore we will develop a method for a direct measurement of imatinib and N-desmethyl imatinib concentrations in the target cells of peripheral blood and bone marrow. This method will also become a basis for individual determination of imatinib uptake into target cells of patients before and immediately after the beginning of therapy. Our aim, therefore, is to validate whether it is possible to determine imatinib and N-desmethyl imatinib concentrations in the blood cells with a highly sensitive LC-MS-MS analytical method and whether the method is applicable in the determination of active drug uptake in the peripheral blood mononuclear cells by OCT1 prior to therapy. By doing so we can provide a clinically useful method for individual determination of OCT1 activity in the target cells, which is important for the success of CML therapy with imatinib.
The relationship between the polymorphisms of the selected genes and concentrations of imatinib and N-desmethyl imatinib in blood plasma as well as their intracellular concentrations will be investigated and the data will be correlated to the treatment outcomes in the population of Slovenian patients. The relationship between the expression of relevant genes for drug transporters and metabolic enzymes in the leucocytes from the bone marrow and those in the peripheral blood will also be evaluated. Specifically, we are interested in the expression of these genes in leucocytes from bone marrow and peripheral blood and its influence on the intracellular concentrations of imatinib and N-desmethyl imatinib. Finally, the expression of the above-mentioned genes will be followed during the therapy at several control time-points. This way, a correlation between the selected gene expression and effectiveness of the treatment can be established.
We believe that the knowledge gained and the methods developed will contribute to the early determination of the optimum imatinib dose, or when necessary, the patient can be prescribed the second line agents, which do not depend on OCT1 activity, to achieve the treatment effectiveness as early as possible.
The role of genetic polymorphisms in treatment result and occurance of side effects in children with cancer
Head: Assist. prof. Janez Jazbec
Although an enormous progress was achieved in the survival rate of childhood cancer, still in some 40 % of all cases the outcome is fatal. A possible way of further improvement is individualization of treatment protocols by distinguishing patients with better tolerance of high dose chemotherapy from those who are more prone to severe toxic events in advance.
Vincristine, a chemotherapeutic agent, is widely used in combination with other agents in the treatment of pediatric haematological malignancies and solid tumors. The most established mechanism by which vincristine inhibits tumor growth is its interference with mitotic spindle microtubules resulting in inhibition of mitosis. Patients treated with vincristine predictably develop peripheral neuropathy which is a major dose-limiting side effect of vincristine. In children treated with identical treatment protocols containing vincristine, the severity of peripheral neuropathy varies greatly. Peripheral neuropathy may be related to polymorphisms in genes involved in vincristine pharmacokinetics or pharmacodynamics: metabolising gene or vincristine toxicity related genes.
Every year we diagnose around 60-70 new patients with childhood cancer in Slovenia. In two years we expect to include around 90 patients with vincristine regimen in their treatment protocol. In all patients we will monitor all possible acute toxicities. In our study we will stratify patients based on signs of standardised clinical neurological examination and parameters of standardized electrophysiological examination according to the severity of vincristine induced peripheral neuropathy. We will genotype DNA from mononuclear blood cells for polymorphisms of vincristine metabolising gene (CYP3A5) or vincristine toxicity related genes (MDR-1 and MAPT).
Based on the results of genetic polymorphism of target genes we expect that we will be able to predict the risk for development of peripheral neuropathy in children treated for cancer. These results may reveal subpopulation of patients with increased risk for peripheral neuropathy after the treatment with vincristine. We could also define group of patients, which may tolerate higher doses of Vincristine in whom intensification of vincristine chemotherapy regime may improve survival of children treated for cancer. The final perspective of all pharmacogenetic studies is therapy individualization. By finding a patient-specific “optimal” dose, we could balance the risk of relapse and the risk of severe adverse effects more precisely.
Development and evaluation of radiolabelled bio-molecules for targeted radionuclide therapy and imaging of neuroendocrine tumours
Head: Assist. prof. Jurij Fettich
Number of cancer and cancer deaths in western countries continuously increases over the past few decades. The fact that it is the third leading cause of deaths in the developed countries, makes cancer a major research challenge over the past two decades. Use of systemic chemotherapy agents for treatment in systemic disease is often limited by serious side effects to normal tissues and lack of their selectivity. Sub-optimal doses result in therapeutic failure and the development of drug resistance. Tumour-targeting therapeutics, based on bio-molecules such as peptides and antibodies, are becoming increasingly important in therapy regiments due to their high selectivity and relative low toxicity to normal tissues. When such a bio-molecule is used as a vector for delivery of radioactive isotope to affected tissue, its therapeutic power is greatly improved because of radiation dose, specifically directed to cancer cells.
Targeted molecular radiotherapy is a new therapeutic modalitiy for patients with inoperable neuroendocrine tumours, who are not suitable chemotherapy. Indications for treatment with radiolabelled octreotide analogs are: inoperable, metastatic neuroendocrine carcinomas with high somatostatin expression confirmed by diagnostic octreotide scintigraphy, normal bone marrow reserve (Hb ) 10g/l, WBC ) 3.0*109/l, platelets ) 100*109/l), adequate renal function and good performance status. Majority of neuroendocrine tumours express receptors for somatostatin on their cell membrane. Besides somatostatin, also its analogs, like octreotide can bind to the receptors.
For imaging of neuroendocrine tumours in Slovenia radiolabelled 99mTc labelled somatostatin analog (hydrazinonicotinyl-[D-Phe1, Tyr3]-octereotid - (99mTc-HYNIC-TOC) is used since 2002. In 2006-2009 University medical centre in Ljubljana has implemented targeted molecular radiotherapy of neuroendocrine tumours with radiolabelled analogs 1,4,7,10-tetraazocyclododekan-1,4,7,10-tetraacetic acid-[D-Phe1, Tyr3]-octreotid (90Y-DOTATOC) and 177Lu-1,4,7,10-tetraazocyclododekan-1,4,7,10-tetraacetic acid-[D-Phe1, Tyr3]-octreotate (177Lu-DOTATATE).
In cell biology there is a well konwn sindrom, where number of available binding sites on a celll membrane is decreased – so called down-regulation. The mechanism is not entirely understood, data in the literature shows that amount of the substrate and possible saturation of binding sites can cause the down-regulation. There is not conclusive data on amount or concentration of peptide, where down-regulation of somatostatine receptors occurs. It is postulated that if the concentration of peptide on affected sites is too high, accumulation of radiolabelled peptide is diminished resulting in decreased sensitivity of imaging procedure or sub-optimal succes of treatment of neuroendocrine tumours.
The basis of this proposal is optimisation of targeted molecular radiotherapy of patients with neuroendocrine tumours using radiolabelled octreotide analogs. We will study influence of amountg of total peptide mass and receptor binding sites on accumulation of radiolabelled octreotide analog on somatostatin receptor. Further on we will investigate the effect of classical therapy with longacting somatostatin analogs on efficiency of imaging of somatostatine receptors using 99mTc-HYNICTOC and/or targeted molecular radionuclide therapy using radiolabelled analogs 90Y-DOTA-TOC and/or 177Lu-DOTATATE.
FINISHED ARRS PROJECTS
Genetic factors in osteoporosis
Head: Prof. Janja Marc
Cell-free nucleic acids in diagnosis of coronary atherosclerosis
Head: Assoc. prof. Darko Černe
Genetics of breast cancer and hormone replacement therapy
Head: Prof. Ksenija Geršak
Thiopurine pharmacogenetics - novel diagnostics and individualized therapy
Head: Prof. Irena Mlinarič Raščan
Analysis of gene expression associated with apoptosis using microarrays
Head: Prof. Irena Mlinarič Raščan
Determination of surface properties of solid and semisolid materials by solid and semisolid materials with inverse phase gas chromatography
Head: Prof. Stane Srčič
Pharmacogenetics of 6-mercaptopurine and thiopurine methyltransferase
Head: prof. dr. Irena Mlinarič Raščan
Preparation and evaluation of stabilised control blood for haematology analysers in Slovenian external quality assessment scheme
Head: Assist. prof. Milan Skitek
2011-2013 EPTA: EP4 as terapeutic target. French –Slovenian bilateral project. CEA-ARRS project.
FINISHED INTERNATIONAL PROJECTS
2010-2011 MAL: Modulation of Apoptosis in Lymphoma. French–Slovenian bilateral project. CEA-ARRS project.
2006-2007 INOMA: In the search of novel modulators of apoptosis. French–Slovenian bilateral project. CEA- MVZT Project.