7^th Global Experts Meeting on Neuropharmacology July 30-31, 2017 Milan, Italy

Eduard H Panosyan is a Pediatric Hematologist-Oncologist, who as a Physician-Scientist developed his initial research expertise in studying amino acid metabolism in childhood acute lymphoblastic leukemias. His further research skills are related to studies of pediatric brain tumors. Consequentially, as an Independent Investigator, he has developed research interests on metabolic pathways and related cellular mechanisms by which brain tumor cells sustain malignant proliferation in vitro and in vivo. Main focus of his investigations is on augmented metabolism of amino acids promoting progression of malignant brain tumors. Identification of druggable targets for heterogeneous metabolic patterns in glioblastomas and medulloblastomas may allow effective therapeutics development. His lab is on the verge of conducting exciting pre-clinical experiments to test some of the anti-metabolites with chemotherapy against intracranial brain tumors.

Statement of the Problem: Brain tumors cause major morbidity/mortality. Glioblastoma (GBM) in adults and medulloblastoma in children are major types of clinically aggressive high-grade CNS malignancies. These have increased uptake of radio-labeled amino-acids (AA) used for PET scans. Central amino-acid Glutamine (Gln) is crucial for growth of brain tumors. Nevertheless, there aren’t clinically effective pharmaceutical interventions against AA metabolism of brain tumors. In contrary, anti-metabolites like bacterial enzyme asparaginase (ASNase, which effectively depletes asparagine and Gln in blood and CSF) are successfully used to treat acute leukemias. Thus, we aimed to study if manipulation of AA metabolism may have a role in developing therapeutics against brain tumors. Methodology & Theoretical Orientation: We conducted series of analyses on large brain tumor databases containing molecular signatures and clinical outcomes. In addition, using ASNase as a research tool, we carried out series of pre-clinical investigations on glioma cell-lines and on mice that harbor medulloblastomas. Findings: We have shown that lower co-expressions of BCAT1, ASNS and GLS are associated with better outcome in 66 newly diagnosed GBM patients (UCLA-probesetanalyzer database). Three additional GBM datasets (r2.amc.nl) showed that at least 4 additional AA-related enzymes-which have higher expression in GBM, also predict poor outcome in 504 GBM patients (TCGA database). Several of these enzymes have differential expression in pediatric brain tumors, including subtypes of medulloblastomas. Mouse models of medulloblastomas demonstrated anti-tumor efficacy of E. coli-ASNase+TMZ against DAOY-SQ model and of Erwinia Chrysanthemi-ASNase, which shows preliminary efficacy in SMO/SMO spontaneous medulloblastoma model. All ASNase formulations showed in vitro activity against glioma cells with variable IC50s. Conclusion & Significance: Our studies support the concept that AA metabolism plausibly harbors targetable nodes, which can be studied for new anti-metabolite therapeutics development for brain tumors. They provide opportunities for combined therapies due to less myelosuppressive toxicity profile.

Alexandra Kasabova received his Master’s degree in Faculty of Pharmacy at Medical University–Sofia, in November 2014. She is a PhD student at the Department of Pharmacology, Pharmacotherapy and Toxicology, Medical University-Sofia from November 2015 until now. She has interests in the field of neuropharmacology and has experimental works on neuronal cell line SH-SY5Y and synaptosomal fraction in normal state and in condition of 6-OHDA–induced oxidative stress

Certain asymmetrically substituted xanthines (propentofylline) are used to treat CNS disorders such as dementia in Alzheimer's disease. In the present study, we investigated the toxicity of 14 newly synthesized derivatives of caffeine-8-α-methyl thioglycolic acid (Jα-0 to Jα-13) on human neuroblastoma derived cells in vitro. The protective effects of the less cytotoxic compounds were also studied in 6-hydroxydopamine (6-OHDA) induced oxidative stress in neuroblastoma SH-SY5Y cells. To evaluate the effects of the treatment, SH-SY5Y cells viability (measured by MTT-test) was used as markers of oxidative damage. SH-SY5Y cells are often used for studying the processes of neurotoxicity and neurodegenerative diseases, such as Parkinson’s. The cells possess the ability to synthesize dopamine and express dopamine transporter (DAT), a protein expressed only in dopaminergic neurons within the central nervous system. Administered alone, all compounds revealed statistically significant toxic effects, compared to the control (untreated cells). Three of the compounds, Jα-6, Jα-7 and Jα-9, had lower neurotoxic effects: Jα-6 decreased cell viability by 10 %, Jα-7 –by 15% and Jα-9–by 14% (vs. control). All other compounds showed higher cytotoxicity towards SH-SY5Y, decreasing cell viability in the range of 18% to 29%. In a model of 6-OHDA-induced oxidative stress on SH-SY5Y only Jα-7 and Jα-9 (100 μM) revealed statistically significant neuroprotective effects, shown by preservation of the cell viability. In particular, Jα-9 preserved the cell viability by 150% and Jα-7– by 91%, compared to 6-OHDA. Jα-6 had no statistically significant protective effect, compared to 6-OHDA.

Borislav Angelov is a 4th year Pharmacy Student in Medical University of Sofia. Although he graduated in Language High School, he has always been interested in Medical Science, especially Pharmacology and Toxicology. He was accepted into University with impressive results. The medical school offers him great opportunities to develop his interests. During the last few years, he has taken part in various activities related to pharmacy and he has also received certificates from different scientific symposiums and seminars. His experience in the field of pharmacy increases with his new work and research in Neuropharmacology.

Oxidative stress is connected with the pathogenesis of many diseases like neurodegenerative disorders (Parkinson`s and Alzheimer`s disease), atherosclerosis, diabetes and cancer. Oxidative stress leads to disabilities, because free radicals damage a number of structures such as lipids, proteins and DNA. Oxidative stress plays an important role in physiological adaptation and regulation of the signaling cellular transduction as well. The treatment of isolated rat brain synaptosomes with 6-OH-dopamine (6-OHDA) is a convenient in vitro sub-cellular system for the investigation of processes, which play role in the neurodegenerative disease, including Parkinson’s and Alzheimer’s diseases. The mechanism of 6-OH-dopamine neurotoxicity includes the formation of ROS and reactive metabolites, as a result of its metabolism in mitochondria of the nerve cells. In this study, we investigate the effects of 6 newly synthesized caffeine derivatives on isolated rat synaptosomes. The main parameters: Synaptosomal viability and GSH depletion, determined in the model of 6-hydroxydopamine (6-OHDA)-induced toxicity (a model of oxidative stress), were investigated. In conditions of 6-OH-dopamine-induced oxidative stress (at concentration 150 μM) on isolated rat synaposomes, all 6 compounds (at concentration 100 μM) revealed statistically significant neuroprotective effect by preservation of synaptosomal viability, measured by MTT-test and GSH deletion. The compound 4d showed statistically significant higher neuroprotective and antioxidant activity in this model, compared to the other 5 compounds.

Martin Vasilev is a final year Pharmacy Student in Medical University of Sofia. Having an interest in wide range of health areas, he has a Clinical Pharmacy specialization, a practice in Hospital Pharmacy and a number of certificates from scientific symposiums, practical trainings and seminars. During his education, he gained experience in creating and introducing a variety of scientific oral presentations in different subjects. With his last work in the area of Neuropharmacology, he developed his skills in such an attractive and advanced method like wire myography. Working together with the Bulgarian Academy of Science, he presents one significant research which expands his growing interest in the Neuroscience. Exploring this field, he hopes to continue working on other scientific projects.

Caffeine, the most widely consumed psychoactive substance in the world is contained not only in coffee but also tea, chocolate and a variety of drugs, including appetite suppressants, diuretics, analgesics and decongestants. Other substances with similar structure to that of the alkaloids present in the coffee, are an important component for the development of a potential treatment of Alzheimer's disease, asthma, cancer, diabetes and Parkinson's disease. We set to evaluate some newly synthesized derivatives of caffeine thioglycolic acid and their influence on contractility of brain vessels (a. basilaris). Two of the compounds, JTA3 and JTA5, derivatives of caffeine-8-thioglycolic acid, were investigated for possible vasoactive effects on brain vessels (a.basilaris). Arterial segments with length of 1.8-2 mm were mounted and tested on dual wire myograph (model 410A, JP Trading, Denmark). Intact basilar artery segments, JTA3 and JTA5 showed opposite physiological effects of caffeine-an increase of vascular tone. These effects may occur due to the different structure of the molecules of these caffeine derivatives and their different affinity to adenosine receptor subtypes (A1 or A2).

P Banerjee has expertise in cellular assays for evaluating and screening pharmacological compounds stems from her thorough training in cancer biology and signal transduction mechanisms. During her earlier work for PhD, she worked with geriatric human patients to assess the efficacy of a phase IV medication for alleviating the biochemical signatures of senescence. Later she has identified the key signaling pathway of presumed antioxidants that paradoxically triggered oxidative stress, particularly in fast-growing cancer cells. Her most recent work extends to structural biology and aims to identify and characterize compounds that target a novel interfacial binding pocket in the dimeric GPCR, smoothened (Smo), mutation of which is causally associated with medulloblastoma in humans.

Small molecules targeting the transmembrane domain (TMD) of the smoothened (Smo) receptor, the main transducer of the Hedgehog (Hh) signaling pathway, are in clinical trials for the treatment of basal cell carcinoma and medulloblastoma, which is one of the most aggressive brain tumors in childhood. The clinical efficacies of these inhibitors are hampered by the occurrence of point mutations located at the TMD binding site leading to resistance. Thus, identification of novel drugs with original pharmacological properties and targeting regions of the Smo protein exhibiting few mutations, would provide a new strategy for potent cancer therapies. Multiple genetic, biochemical and crystallographic data suggest that Smo functions as a homodimer. We have now identified two potentially druggable cavities at the interface of the homodimer that have been screened in silico to identify putative dimer-biased antagonists. From an in-house library of 5 million commercially drug-like compounds, we selected and purchased 64 compounds that we have tested for their inhibition properties using already developed cellular assays aimed at measuring Hh-induced activation through Smo. Among the positive hits identified, and after their in house synthesis, one compound (DR90) inhibited mouse cerebellar granule cell proliferation induced by Hh (IC50=2.5 µM). DR90 was found to be an allosteric competitor of the Smo agonist SAG towards this response. Binding experiment analysis of DR90 to wild type human Smo receptor and to various mutants using [3H] MRT-92 and bodipycyclopamine indicated a non-conventional mode of binding for DR90. Further experiments are in progress to identify the binding mode of DR90 and related molecules to Smo. Our studies should help to design more effective therapeutic strategies for treating Hh-linked cancers and associated chemoresistance and better understanding the transduction mechanism of Smo, an atypical member of the G protein-coupled receptor superfamily.

Dorina Petrova is a Neurology Specialist with experience in the Neurotology and Oto-rhino-laryngology. She has wide experience in clinical practice with peripheral and central vestibular diseases. She performs research in fields of early diagnosis of cerebrovascular diseases, prevention and treatment of diseases of the peripheral nervous system as diabetic polyneuropathy, applying of innovative pharmacological and physical methods of diagnostics and treatment.

Background: The maintenance of posture involves complex mechanisms integrating information derived from the visual, vestibular and somatosensory systems. Malfunction of one of these sensory systems leads to disequilibrium. Diabetic polyneuropathy (DPN) is a common complication of diabetes mellitus provoked by a long-term hyperglycemia leading to induced and increased production of free oxygen radicals, which cause disturbance of the sensory afferent and efferent pathways and lead to nerve damage and diminution of postural stability during standing and walking. Alpha-lipoic acid (ALA) is one of the most effective antioxidant kill free radicals that help lower blood sugar levels. The aim of the present study is to assess the postural stability of patients with type 2 diabetes mellitus and DPN after treatment with combined therapy of ALA and benfotiamin, pyridoxine and cyancobalamine together, by static posturography. Patients & Methods: 60 patients randomly divided in two groups took part in this study. The first group was treatment with 600 mg ALA and the second–with combined therapy. The postural stability was measured in four conditions– stance on stable and on foam surface with open and closed eyes. The displacements of center of foot pressure in both medio-lateral (ML) and anterior-posterior (AP) directions were registered and the mean amplitude and mean velocity of postural sways were analyzed. The assessments were made on the first, 6th and 60th day after the drug therapy. Results: No patient under therapy with deterioration. Patients with combined therapy showed considerably higher improvement than the other group. The improvements of mean sway velocities were higher than the ones for sway amplitudes in all experimental conditions. The best effect was observed for velocities of postural sways of patients with combined therapy. Conclusion: Treatment with combined therapy showed stabilizing effect on the posture that leads to improvement of the quality of life of patients with type 2 diabetes mellitus and DPN.

Applied pharmacologist, European specialist in lab medicine , independent researcher

In jurisdictional situation one most important milestones is to determinate when a fact is voluntary and when not to determinate the entity of the legal penalty in some crime situations. A deep knowledge in pato-phisiological psycology and neurology can help the judge in this specific Contest to be added to the neuroscientist expert official opinion .(1) The same this information can be used in order to reconsider old crime process in the right light or for Insurance or any other interested in. To have a really objective method can help in this process. Discussion- conclusion In order to improve the knowledge in some crime behavior we think is usefull consider the Physiopathology of amygdala and related systems whit all science methods aviable today. “In activation of amygdala the response is more rapid than when involved the frontal cortex “(2) This publication has not any therapeutic or diagnostic intent only to produce scientific hypotesys.