Abstracts Book

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Abstracts Briefings

SESSION 1: 
New in vivo tools in Pharmacology: CRISPR/Cas and conditional mice

Speaker: Lluís Montoliu
New animal models with CRISPR tools

Genome editing has boosted our capacity to produce numerous animal models, for investigating in vivo the cause of altered gene expression and its resulting pathological consequences. Now, with the use of CRISPR genome editing tools, a large variety of genome-edited mice, zebrafish and other useful animal models, can be created easily and rapidly, and at a fraction of the cost that used to be limiting for the generation of transgenic and knockout mice using classical techniques. Single base pair insertions, deletions, substitutions, larger deletions can readily be made with CRISPR reagents directly on mouse embryos, without requiring the use of embryonic stem cells. Reproducing patient-specific mutations in the homologous mouse gene counterparts is now feasible and it is perhaps one of the most powerful benefits of genome editing techniques for the production of in vivo models. Other more challenging projects, including knockin developments, are still difficult but doable with this new technology. In this presentation, I will review the current status of the CRISPR revolution in what it concerns for the generation of new animal models for biomedical research. I will illustrate my talk with numerous examples of mouse models of rare diseases, such as albinism, the genetic condition we have been studying in the lab for many years.

Speaker: Sagrario Ortega
Conditional Mouse Models, Strategies and Applications

The laboratory mouse is widely considered the model of choice to study gene function and regulation and the best suited for modeling human disease. The genetic modification of the mouse germ line, achieved for the first time in the last quarter of the 20th century when animal transgenesis was first established, has substantially progressed along the last 20-30 years. The development of gene-targeting technologies based in the use of mouse embryonic stem cells and more recently the new tools for genome editing, especially the CRISP/Cas system, have facilitated the targeted and precise modification of the mouse genome. The introduction of virtually any kind of genetic modification including gene deletions, point mutations, tag or reporter insertions, gene replacement and even chromosomal translocations in the mouse genome is currently relatively simple. However, constitutive genetic modification, in all the cells of the animal and at all stages of development, has many limitations including, in many cases, embryonic lethality that prevents the use of this strategy for modelling disease or studying gene function in the adult. Conditional models, in which the genetic modifications are controlled temporally and spatially have overcome these and other limitations.

Two of the most widely used strategies for conditional genetic manipulation in mammals are exported from prokaryotic organisms. The tetracycline inducible system, used by E.coli strains carrying the Tn10 transposon to control tetracycline resistance, was initially adapted by Mosen and Bujard to control gene expression in eukaryotes. The system, based in the use of a tetracycline responsive promoter to drive gene transcription, has evolved and has been optimized so that we have many tools today to use it in mouse models for inducing or repressing expression of any given gene. While the tetracycline system controls gene transcription and, therefore, is an epigenetic modulator, another conditional system, based in the use of the Cre recombinase of the E. coli bacteriophage P1, induces site-specific recombination by binding and recombining two target DNA sequences of 34 nucleotides called loxP sites. The loxP sites are not present in the mammalian genome but can be introduced by gene targeting or gene editing to create “primed”  alleles, called floxed alleles, that behave as wild type ones but are susceptible of Cre-mediated recombination to be converted to knockout (loss of function) or knockin (precise mutations) alleles. Cre may also catalyze “in trans” the targeted integration of a loxP containing transgene into a pre-defined position in the genome where a loxP site has been previously introduced or even precise chromosomal translocations. The Flp recombinase from S. cerevisiae works in a similar way to Cre, recombining two frt target sites whose structure is similar to loxP sites but have different sequence, so that both systems can be used in the same mouse simultaneously. Fusions of these recombinases with the ligand-binding domain of the estrogen receptor (ER), previously modified (ERT2) to reduce its affinity for the endogenous 17b estradiol and specifically bind to 4-OH tamoxifen (4OHT) allows temporal control of Cre or Flp activity. These fusions are retained in the cytoplasm of the cells, bound to the HSP90 chaperon protein and are released and internalized in the nucleus upon 4OHT administration.

Several examples of the use of these systems for studying gene function, tracing cell fate during development, modelling cancer and identifying new potential targets for cancer therapy in the mouse will be presented.

Two of the most widely used strategies for conditional genetic manipulation in mammals are exported from prokaryotic organisms. The tetracycline inducible system, used by E.coli strains carrying the Tn10 transposon to control tetracycline resistance, was initially adapted by Mosen and Bujard to control gene expression in eukaryotes. The system, based in the use of a tetracycline responsive promoter to drive gene transcription, has evolved and has been optimized so that we have many tools today to use it in mouse models for inducing or repressing expression of any given gene. While the tetracycline system controls gene transcription and, therefore, is an epigenetic modulator, another conditional system, based in the use of the Cre recombinase of the E. coli bacteriophage P1, induces site-specific recombination by binding and recombining two target DNA sequences of 34 nucleotides called loxP sites. The loxP sites are not present in the mammalian genome but can be introduced by gene targeting or gene editing to create “primed”  alleles, called floxed alleles, that behave as wild type ones but are susceptible of Cre-mediated recombination to be converted to knockout (loss of function) or knockin (precise mutations) alleles. Cre may also catalyze “in trans” the targeted integration of a loxP containing transgene into a pre-defined position in the genome where a loxP site has been previously introduced or even precise chromosomal translocations. The Flp recombinase from S. cerevisiae works in a similar way to Cre, recombining two frt target sites whose structure is similar to loxP sites but have different sequence, so that both systems can be used in the same mouse simultaneously. Fusions of these recombinases with the ligand-binding domain of the estrogen receptor (ER), previously modified (ERT2) to reduce its affinity for the endogenous 17b estradiol and specifically bind to 4-OH tamoxifen (4OHT) allows temporal control of Cre or Flp activity. These fusions are retained in the cytoplasm of the cells, bound to the HSP90 chaperon protein and are released and internalized in the nucleus upon 4OHT administration.

Several examples of the use of these systems for studying gene function, tracing cell fate during development, modelling cancer and identifying new potential targets for cancer therapy in the mouse will be presented.

Speaker: María Angeles Moro
Investigation of Rodent Adult Neurogenesis in Homeostasis and Disease: An example of the use of Conditional Mice in Neuropharmacology

The conditional gene knockout method is a very ingenious strategy based on the elimination of a specific gene in a certain cell type. It is of great utility for the study of the function of genes in selected cell types. It is also valuable in the study of the relevance of cellular processes which depend upon the gene which is conditionally knocked-out. In this presentation, we will review how conditional knock-out mice can provide essential information in the study of the function of specific receptors in adult neurogenesis in the healthy brain, and also how these mice can be applied to explore the role of this process in the injured brain. 

Opening Lecture: 
 Stop the Mast Cells and the Eosinophils by any means

Speaker: Francesca Levi-Schaffer
Stop the Mast Cells and the Eosinophils by any means!

The pivotal effector cells of allergic inflammation are the mast cells and the eosinophils. Mast cells, as activated by IgE mechanisms via allergens, are the recognized primum movens while eosinophils infiltration and persistence in the inflamed tissue with the mast cells are the accepted features of the late stage and of the chronic outcome of allergy.

During the years we have defined a pro-inflammatory cross-talk between these two cells that we have named the Allergic Effector Unit (AEU). We found that mast cell/eosinophil interactions result in increased eosinophils chemotaxis, survival, degranulation, cytokine production and in mast cell survival, IgE-dependent and independent degranulation and cytokine production. These effects are mediated by both released mediators (soluble interactions) and by receptor/ligands binding (physical interactions). Prominent players of the activating “physical” AEU are the two activating receptors (ARs)/ligands CD48 and 2B4. Nevertheless, we have also described the presence and functional activity of two inhibitory receptors (IRs), i.e. CD300a and Siglec-7, on mast cells and on eosinophils that can indicate a possible anti-inflammatory or even pro-resolution activity within the AEU and globally as mediated by mast cells and by eosinophils.

The goal of our research is to define potential new targets for immunopharmacological intervention in allergic diseases by blocking ARs, i.e. CD48, or by activating IRs, i.e. CD300a and Siglec-7. We indeed found that CD48 is significantly upregulated on human and murine asthma on mast cells and eosinophils and in the presence of S. aureus, the prominent bacteria infecting atopic tissues. We have therefore studied CD48 modulation in vitro and in vivo and the outcome of its blockade and found that CD48 is a key player in allergic diseases. Similarly, we have found that CD300a and Siglec-7 are expressed by eosinophils and mast cells of allergic patients and described their role in downregulating these cell functions.

Thus, our strategy is to treat allergy by inhibiting activation and/or by activating inhibition of mast cells, eosinophils and the AEU. Translationally this strategy will have to take into account the allergic patient endotype.

Plenary Lecture: 
 Pending Information

Speaker: Nathalie Vergnolle
Protease-Activated Receptors (PARs) in inflammation and pain: what should we target?

The pivotal effector cells of allergic inflammation are the mast cells and the eosinophils. Mast cells, as activated by IgE mechanisms via allergens, are the recognized primum movens while eosinophils infiltration and persistence in the inflamed tissue with the mast cells are the accepted features of the late stage and of the chronic outcome of allergy.

During the years we have defined a pro-inflammatory cross-talk between these two cells that we have named the Allergic Effector Unit (AEU). We found that mast cell/eosinophil interactions result in increased eosinophils chemotaxis, survival, degranulation, cytokine production and in mast cell survival, IgE-dependent and independent degranulation and cytokine production. These effects are mediated by both released mediators (soluble interactions) and by receptor/ligands binding (physical interactions). Prominent players of the activating “physical” AEU are the two activating receptors (ARs)/ligands CD48 and 2B4. Nevertheless, we have also described the presence and functional activity of two inhibitory receptors (IRs), i.e. CD300a and Siglec-7, on mast cells and on eosinophils that can indicate a possible anti-inflammatory or even pro-resolution activity within the AEU and globally as mediated by mast cells and by eosinophils.

The goal of our research is to define potential new targets for immunopharmacological intervention in allergic diseases by blocking ARs, i.e. CD48, or by activating IRs, i.e. CD300a and Siglec-7. We indeed found that CD48 is significantly upregulated on human and murine asthma on mast cells and eosinophils and in the presence of S. aureus, the prominent bacteria infecting atopic tissues. We have therefore studied CD48 modulation in vitro and in vivo and the outcome of its blockade and found that CD48 is a key player in allergic diseases. Similarly, we have found that CD300a and Siglec-7 are expressed by eosinophils and mast cells of allergic patients and described their role in downregulating these cell functions.

Thus, our strategy is to treat allergy by inhibiting activation and/or by activating inhibition of mast cells, eosinophils and the AEU. Translationally this strategy will have to take into account the allergic patient endotype.

Known proteases such as thrombin or trypsin cleave PARs at established sites on the extracellular domain of the N-terminal domain of the receptors to induce specific intracellular signals. However, a growing number of other proteases have been identified and cleave PARs at divergent sites, activating distinct signaling pathways. With this concept of biased agonism, the pharmacology of these receptors has revealed to be more complicated than anticipated. A better understanding of the nature and specificity of the proteases present in physiological and pathophysiological conditions is therefore urgently needed. This lecture will focus on presenting the pharmacology of PARs, their involvement in inflammation and pain mechanisms, and will reflect on how we should consider possible therapeutic interventions targeting PARs or other members of the proteolytic balance. 

SESSION 2: 
New targets in cardiac and cardiovascular disorders

Speaker: Thomas Wieland
Targeting Histidine Phosphorylation by Nucleoside Diphosphate Kinases in Cardiovascular Disorders
Speaker: Ricardo Caballero
Recent Advances in the Pharmacological Treatment of Cardiovascular Disease

Cardiovascular disease (CVD) is a group of disorders of heart and blood vessels that includes among others: hypertension, coronary artery disease, heart failure, cerebrovascular disease, and peripheral vascular disease. CVD is the leading cause of death in the world: more people die annually from CVD than from any other cause. During the last decades there were huge advances in the pharmacological treatment of some of these diseases. Nowadays, drugs for the treatment of hypertension or coronary artery disease are very efficacious, safe, and cheap. Furthermore, in recent years new anticoagulants, hypolipidemic and antidiabetic drugs, or drugs for the treatment of some forms of heart failure have been developed and successfully used in many patients. However, pharmacology of CVD is not devoid of remarkable failures and mistakes at all stages of drug development. In this regard, there are some examples of drugs with serious concerns about their efficacy and/or safety. Furthermore, even after extensive basic and clinical research in the field the pharmacological treatment of some severe forms of CVD such as acute heart failure or heart failure with preserved ejection fraction is clearly suboptimal. To circumvent all these challenges, the use of novel strategies and tools is necessary in order to improve the development of new drugs for treating CVD patients. 

Speaker: Mattijs Blankesteijn
Interventions in WNT Signaling Ctimulate Cardiac Regeneration

Activation in WNT signaling has been identified in many cardiovascular diseases, but its potential implications for therapeutic intervention are only beginning to be understood. Several studies from different research groups have shown, however, that pharmacological inhibition of WNT signaling after myocardial infarction reduces scar size and improves cardiac function. A plausible explanation for this is the induction of newly formed cardiomyoyctes in the border zone of the infarct. Given the importance of WNT signaling in the control cell differentiation and stem cell biology it is tempting to propose WNT inhibition as a mediator of cardiac regeneration.

Speaker: Aida Collado (Oral Communication)
Systemic Inflammation is Reduced in Primary Hypercholesterolemia Patients after an Oral Fat Load Administration

Activation in WNT signaling has been identified in many cardiovascular diseases, but its potential implications for therapeutic intervention are only beginning to be understood. Several studies from different research groups have shown, however, that pharmacological inhibition of WNT signaling after myocardial infarction reduces scar size and improves cardiac function. A plausible explanation for this is the induction of newly formed cardiomyoyctes in the border zone of the infarct. Given the importance of WNT signaling in the control cell differentiation and stem cell biology it is tempting to propose WNT inhibition as a mediator of cardiac regeneration.Introduction and Objectives: Primary hypercholesterolemia (PH) is a lipid disorder characterized by elevated levels of cholesterol and low-density lipoprotein (LDL). Low-grade systemic inflammation is associated with PH, which might explain the higher incidence of cardiovascular diseases. In this study, we have evaluated the effect of an oral fat load (OFL) on different immune parameters and its consequences in PH patients.

Methods: 22 PH patients’ whole blood was analyzed by flow cytometry to determine platelet activation (P-selectin+ and PAC-1+), leukocyte activation (CD11b+ and CD69+) and the percentage of circulating platelet-leukocyte aggregates before and 4h after a treatment with a commercial liquid preparation of long-chain triglycerides (ω6/ω3 ratio >20/1, Supracal, Nutricia). The parallel-plate flow chamber was employed to study platelet-leukocyte and leukocyte adhesion to the arterial endothelium. Plasma inflammatory markers were determined by ELISA.

Results: Four hours after OFL, PH patients presented a lower percentage of activated platelets. The percentage of circulating eosinophils, type 1 monocytes, platelet- eosinophil, but not platelet-neutrophil aggregates was significantly decreased. Surprisingly, the percentage of regulatory T lymphocytes (Treg) was significantly increased. Moreover, a tendency in reduced percentages of platelet-monocyte (types 1 and 2) aggregates was also observed. Leukocyte adhesion to the dysfunctional endothelium (TNFα-stimulated) was significantly ameliorated together with plasma levels of CXCL4, sP-selectin, CXCL8, CCL2, CCL5 and TNFα.

Conclusion: After 4h OFL, PH patients have a decreased platelet activation state, circulating levels of different chemokines and soluble adhesion molecules as well as diminished platelet-leukocyte and leukocyte adhesion to the dysfunctional arterial endothelium. Therefore, OFL may become a powerful tool to dampen the systemic inflammation associated with PH and the development of further cardiovascular events. Funding: This work was supported by the Spanish Ministry of Economy and Competitiveness [grant numbers SAF2014-57845-R, SAF2017-89714-R]; Carlos III Health Institute and the European Regional Development Fund [grant numbers PI15/00082, PIE15/00013, PI18/00209].

Speaker: Néstor De La Visitación (Oral Communication)
Probiotics Prevent Hypertension in a Murine Model of Systemic Lupus Erythematosus Induced by TLR7 Activation

Objective: We tested whether Lactobacillus fermentum CECT5716 (LC40) and/or Bifidobacterium breve CECT7263 (BFM26) prevent hypertension, endothelial dysfunction and intestinal dysbiosis in an inducible model of systemic lupus erythematosus (SLE).
Materials and methods: Eight-week-old BALB/cByJRj mice were treated with 1.25 mg of the agonist of TLR7 Imiquimod (IMQ) for 8 weeks. At the same time, LC40 (109 UFC/mL) and BFM26 (109 UFC/mL) were gavaged daily.
Results: IMQ induced intestinal dysbiosis characterized by both reduction in the Firmicutes/Bacteroidetes ratio (F/B) and α-diversity measure by Chao-richness and numbers of species. LC40 and BFM26 could not restored theses parameters. The three-dimensional principal component analysis of the bacterial taxa in faecal samples showed perfect clustering among groups (CTR and IMQ). The clusters corresponding to LC40 and BFM26 were more similar to IMQ. The short chain fatty acids (SCFA) producing bacteria were also analyzed, the level of acetate-producing bacteria was found elevated but neither of the probiotics restored it. BFM26 and LC40 were found set in the gut microbiota of animals treated respectively. LC40 and BFM26 treatments significantly reduced lupus disease activity assessed by plasma double-stranded DNA autoantibodies, as well as B cell populations in spleen, which were found elevated in the IMQ group. Both probiotics reduced the expression on IFNγ which was found elevated in the SLE model. Also, LC40 and BFM26 treatments reduced Th17 in mesenteric lymph nodes. However, only in the LC40-treated group Treg were elevated. Aortae from IMQ mice showed reduced endothelium dependent vasodilator responses to acetylcholine, which were normalized by both BFM26 and LC40. Furthermore, vascular ROS contents were increased in IMQ mice and reduced by both probiotic strains.
Conclusion: BFM26 and LC40 prevented the development of hypertension in this model, and normalized the endothelial function.

SESSION 3: 
Parallel Roundtable. Education in Pharmacology

Speaker: Michael Spedding
IUPHAR, Education and Collaborations World-Wide

The International Union of Basic and Clinical Pharmacology (IUPHAR) represents ~37,000 pharmacologists world-wide and is affiliated to WHO for Health Care. The nomenclature committee (current chair Steve Alexander) has structured modern pharmacology and IUPHAR has published 125 publications with an H-Index of >80. NC-IUPHAR organises two meetings/year. Over 90 subcommittees of about 700 expert scientists back our publicly available databases, accessed freely by scientists in 160 countries:

  • The IUPHAR/BPS guidetopharmacology.org, with help from the BPS and Wellcome Trust
  • The guidetoimmunopharmacology.org (see Francesca Levi-Schaffer’s presentation)
  • The IUPHAR/MMV guide to malaria pharmacology.org, financed by the Medicines for Malaria Initiative.

We have also set up, helped by ASPET, CNPHARS, JPS, and HPS, the pharmacology education project website, which is an ongoing project, freely available to all. The IOSP project run by David Lewis has given multiple training courses on animal use and ethics in China, Africa and India. Courses in clinical pharmacology, and on pharmacovigilance have been run in China, India and Africa. We have signed an agreement with Karolinska Institute to progress academic drug discovery and clinical pharmacology, obtaining two seed grants. We have organised 8 meetings worldwide on advances in natural products, with an emphasis on how technological advances can engage evidence-based medicine. The next IUPHAR World Congress on natural products is to be held in Hyderabad December 4th-7th, 2019, hosted by IPS.  An enthusiastic young investigator’s committee is now picking up the IUPHAR baton!    

Speaker: Guillermo Diaz
The DoctoraI Programme in Pharmacology

The Doctoral Programme in Pharmacology was established in the year 2002, with the participation of the Faculties of Chemical & Pharmaceutical Sciences, Medicine, Sciences and the Institute of Nutrition & Food Technology (INTA). From 2003, had received a national accreditation by the National Accreditation Institute (CNA-Chile).

Main features of the Doctoral Programme in Pharmacology

The programme entails a face-to-face, full dedication schedule. Candidates hold undergraduate degrees in chemistry, biochemistry, biology and pharmaceutical sciences, including medicine, dentistry, veterinary medicine and biotechnology. Since 2009, the candidate postulates by an on-line application system (https://postulacionpostgrado.uchile.cl). Successful applicants can apply to a CONICYT Scholarship or to any other National or International Granting Institutions, including support by the Faculty of Chemical & Pharmaceutical Sciences, University of Chile. Formal activities are based on two mandatory courses: Advanced Molecular Pharmacology, and Experimental Pharmacology, which focus on molecular modelling, genomics, pharmacological chemistry and clinical pharmacology. It is mandatory that the doctoral thesis work, must generate at least one article published by an international ISI-indexed Journal, where the candidate as a first author. All graduates from the Program are inserted in Research and Academic Institutions, both in the country or abroad. Several graduates proceed to postdoctoral positions in internationally recognised institutions, either locally, or in Europe and North America. The insertion rate is 100% for the total graduate cohorts.

Academic core

At present, the academic core is composed of 34 faculty members with the highest national and international prestige, selected among associate and full professors from the University of Chile. Based on the expertise of academic Core, six lines of research have been established, which warrant proper competitiveness, i.e. Cardiovascular, Cancer, CNS, Immunology, Antimicrobial and Chemotherapy, and Drug Development. Core have collectively authored a total of about 700 ISI articles in the past last 10 years. The Core has also files and obtained 16 patents reflecting an outstanding record.

Achievements

From 2002, more than 90 PhDs in Pharmacology have graduated from the Program. In the past few years, an average of 10 candidates per year have been accepted in the Program, and at present there are 47 candidates carrying out their thesis work. The demand for admission has also increased substantially, in particular by students from other Latin American countries (i.e. Colombia, Venezuela, Costa Rica, Ecuador, Cuba, Peru), thus presenting the Program as attractive choice in the region. The Doctoral Programme in Pharmacology has substantially prioritized international networking, including the University of Regensburg, Germany, for the development of epigenetics; and the University of Mississippi, USA, for Clinical Pharmacology. There are several agreements in progress with the Universidad Autónoma de Madrid, Spain, focusing on system pharmacology, and UNICESUMAR, Paraná, Brazil, focusing on veterinary.

Speaker: Jose Miguel Doña Rodriguez
Speaker: Mª Carmen Montesinos (Oral Communication)
Combination of innovative teaching techniques in Pharmacology seminar classes

Our main objective, besides promoting critical spirit, was to encourage the study of Pharmacology by relating the subject with real life situations. We proposed to combine two strategies applied successfully in our Department, problem-based learning [1] and peer evaluation, with the production of a video.
Based on a prior project consisting in the critical analysis of pharmacological news accessible through social networks [2], we addressed this strategy to students of Pharmacy Degree naive to the subject of Pharmacology (Year 3). Students were instructed to work in teams of 4 and provide a list of 6-10 internet links of news concerning drugs or treatments related to the year curriculum (Central Nervous System or Immunity) to the supervising teacher, who selected the one to be analyzed. Each team had to perform a critical analysis of the news item regarding its veracity and scientific rigor (supported by scientific evidence), giving an assessment of the interest and potential impact in comparison with the standard treatment or drug of reference. Their analysis was presented in a 5-minute video, which was peer-evaluated using a 5- point rubric during the seminar classes.
In general, the activity was well accepted, although some students preferred more structured conventional activities. Most students rated the activity high or very high in terms of promotion of critical spirit. The usefulness towards the learning of pharmacological concepts or to relate the subject with real life was considered mostly satisfactory. The best valued aspect was the interest of the news items. Among the aspects to improve, the most relevant is time-management, followed by the need of assistance by multimedia service of the university to expedite the viewing of the videos.

[1] Recio MC, et al. EDULEARN11 Proceedings (2011) pp. 2488-2495.
[2] Montesinos MC, et al. Basic Clin Pharmacol Toxicol (2018) 123 (Suppl. 2): 75.

Speaker: Gonzalo Casino (Oral Communication)
Information about drugs and related topics in the Spanish general, economic and professional press

Background News about drugs are relevant for citizens, health professionals and industry, but quantitative and qualitative studies are lacking. This study analyses the evolution, frequency and percentage of information about drugs in the Spanish general, economic and professional press, as well as the most relevant pharmacological topics.

Methods We conducted a content analysis of texts about drugs in 17 Spanish newspapers (13 general, 2 economical and 2 professional) identified in Factiva database for the period 2008-2017. We analysed frequencies, absolute and relative numbers of texts and headlines about drugs, and specifically about 10 pharmacological related topics: clinical trials; preclinical research; adverse effects; cancer; infections; antibiotics; resistances; generics; scientific journals and vaccines.

Results In the 2008-2017 period, the volume of texts about drugs decreased by 10%; the national general press published one article about drugs every two days and the regional one every three days; the economic, three in a week, and the professional, 14. In 10 newspapers analysed for 2017, the most covered topics about drugs (n=2,780) were cancer (n=255; 9.2%) and generics (n=152; 5.5%); the least covered was preclinical research (n=28; 1.0%). The press reported six times more on clinical than preclinical research. The economic press is the one that published the most about clinical (5.6%) and preclinical (2.1%) research. In 2017, the information about drugs represents 0.5% (n=822) of the whole information in the general press (n=178,926); 0.8% (n=468) in the economic press (n=55,800), and 8.5% (n=415) in the medical press (4,856).

Conclusions Information about drugs represents less than 1% of the whole information in general and economic press. The professional press publishes the most articles on drugs, followed by the economic and general press. Clinical research on drugs is more covered than preclinical. Cancer and generics are the most intensive covered topics.

Keywords: press, newspapers, news, drugs

SESSION 4: 
Open innovation in drug discovery

Speaker: Isabel Colomina
Health Care and Impact of Migraine from Patients Perspective

Migraine affects more than 4 million people in Spain, of which 80% are women between 20 and 40 years old. Historically migraine has been associated with a simple headache, but it is not, migraine is manifested by recurrent episodes of moderate or severe headache, throbbing and accompanied by nausea, vomiting, sensitivity to light, sounds or the smells.

What we need the patients and how we want to be treated must be part of the active listening of health professionals. The pharmacological innovation that is coming, restricted to a group of patients, we are sure will improve our quality of life, but in the meantime and for those who cannot benefit from it, we must take into account the impact that this disease has on our lives, in all dimensions: personal, family, work, social or educational.

Speaker: María del Buensuceso Fernández del Pozo
Migraine Pharmacology Evolution

Migraine is a highly disabling disease, with recurrent episodic crises, that could progress in some cases to chronicity, having a significant economic and social cost. Its multifactorial ethiology and genetic influence makes difficult the comprehension of its pathophysiology, which is not entirely clarified yet. Until the 40s the only alternative for treating migraine was the symptomatic treatment with non-specific analgesics for this condition, linking antiemetics if necessary, and with which many patients did not find relief. Ergotic drugs were a first step towards more specific drugs, but their efficacy remain limited and their adverse effects and contraindications are considerable. The introduction in the 90’s of the triptans (5-HT1B/1D agonists), which become the gold standard for the treatment of acute crises, relegates the ergotic drugs to the background. However, many patients doesn´t respond yet to treatment. More recently, the first 5-HT1F receptor agonist, lasmiditan, has been authorized, which opens the family of the ditans and allows treating patients with risk or diagnosis of cardiovascular pathology, in which triptans are contraindicated. FDA has also authorized another small molecule, ubrogepant, CGRP receptor antagonist. Even so, almost half of patients fail to reduce the frequency or intensity of seizures. In these cases of chronic migraine, preventive treatment is indicated. Beta-blockers, antiepileptics and antidepressants have been used to prevent migraine with limited effectiveness. In recent years, advances in the knowledge of the pathophysiology of migraine have led to the recent authorization by the EMA and the FDA of monoclonal antibodies against CGRP (galcanezumab, fremanezumab), and its receptor (erenumab), to which other products, such as eptinezumab and the gepants rimegepant and atogepant, in advanced stages of research can be shortly included.

Other targets of interest are the pituitary adenylate cyclase activating polypeptide (PACAP), with a monoclonal antibody in preclinical assays, and its PAC1 receptor, in relation to which a monoclonal antibody already exists in phase II clinical trials that may give rise to a new family of antimigranous. Transient receptor potential (TRP) channels, linked to the release of neuropeptides, constitute another hypothetical target for the control of migraine that is currently under investigation, as well as acid‐sensing ion channels (ASICs), implicated in pain perception and who are inhibited by AINE, or NOD-like receptor protein 3 (NLRP3) inflammasome related to neuroinflammation.

Speaker: Ayoze González
Physiopathology and epidemiology of Migraine

Migraine is a common disorder, with a prevalence of 10-12%. It is more frequent in women and is an important cause of morbidity and loss of daily function. Currently, the main pathophysiological mechanism of migraine includes interactions of the cerebral cortex, brainstem and trigeminovascular system. This spread cortical depression causes sensitization and hyperexcitation of the trigeminovascular system, which leads to inflammatory pathways and to pain aetiology. Various neuropeptides, such as the P substance or the peptide related to the calcitonin gene (GCRP), regulate the trigeminovascular system. It has been demonstrated that migraine patients present increased GCRP level and that it relates to pain crisis. This opens the door to a new therapeutic target in migraine treatment. 

SESSION 5: 
Young Scientist Forum

Speaker: Michael Spedding
Creativity in Science

Michael Spedding, President, Spedding Research Solutions, Le Vesinet, France; Secretary General IUPHAR.

Michael Spedding profits from a long career in pharma and academia, having:

  • Built two research centres, on time and budget,
  • Been responsible for ~50 preclinical projects and for 11 compounds going into clinical phase 1, linked to 3 on the market,
  • Been on the research committees of three pharmas, seeing almost every way drugs can pass or fail,
  • Built-up the NC-IUPHAR databases (guidetopharmacology.org), and the creation of ~90 subcommittees of 700 experts which have structured modern pharmacology, now secretary general IUPHAR,
  • Published >200 papers, >20 patents, and on the Clarivate highly cited scientists ranking (2013-18),
  • Set up a small research company with a drug having orphan drug designation for an ‘impossible disease’: amyotrophic lateral sclerosis,
  • Yet still managed to ‘have a life’, having been a national class athlete, competed for ~60 years, running >115,000 kms, and a good family life,

This requires considerable management skills, prioritisation of objectives, and having a philosophy of science and life. The talk will communicate these in 20 minutes.

Speaker: María Luisa Ferrándiz
How to become a good University Professor
Speaker: Nuria Godessart
Research after PhD
Speaker: Amadeu Gavaldá
From Science to Industry
Speaker: Eduardo Oliver
How to bring Science to Society: from School to Parliament

SESSION 6: 
New advances in the Pharmacology of Respiratory System

Speaker: Angel Cogolludo
Targeting K+ Channels in Pulmonary Hypertension

Pulmonary arterial hypertension (PAH) is a multifactorial and severe disease. PAH pathobiology involves a sustained pulmonary vasoconstriction, endothelial dysfunction, distal pulmonary vessel remodelling, and inflammation. The reduced lumen of pulmonary arteries can cause increased pulmonary vascular resistance leading to right ventricular hypertrophy, heart failure and premature death. Current pharmacological treatments have improved survival rates but annual mortality from PAH remains high. A better understanding of the molecular mechanisms involved in the pathophysiological processes is essential to identify novel drug targets of potential therapeutic interest. Potassium (K+) channels represent the largest and most diverse group of ion channels, being expressed in all mammalian cell types. In pulmonary artery smooth muscle cells, K+ channels are main contributors in setting membrane potential and hence contraction. The activity of K+ channels is finely regulated by a number of vasoactive factors and, in fact, their activation and inhibition has been shown to contribute to the pulmonary vascular effects of main vasoconstrictors and vasodilators, respectively. Among the different K+ channels functionally expressed in the pulmonary vasculature special attention has been paid to the voltage-gated K+ channel Kv1.5 and the two-pore domain K+ channel TASK-1, whose expression or activity is dysregulated in most forms of PAH. A number of mechanisms leading to this K+ channel dysfunction in PAH have been proposed. In recent years, the identification of mutations in the genes encoding for TASK-1 (KCNK3) and Kv1.5 (KCNK5) as novel causes of PAH demonstrates their crucial role in the onset of the disease. Likewise, Kv7 channels have recently emerged as key players regulating vascular tone. The availability of selective activators currently indicated for other conditions makes Kv7 channels an attractive target in PAH. In summary, targeting K+ channels to increase their activity or prevent their impairment may represent a possible future therapeutic strategy for PAH.

Other targets of interest are the pituitary adenylate cyclase activating polypeptide (PACAP), with a monoclonal antibody in preclinical assays, and its PAC1 receptor, in relation to which a monoclonal antibody already exists in phase II clinical trials that may give rise to a new family of antimigranous. Transient receptor potential (TRP) channels, linked to the release of neuropeptides, constitute another hypothetical target for the control of migraine that is currently under investigation, as well as acid‐sensing ion channels (ASICs), implicated in pain perception and who are inhibited by AINE, or NOD-like receptor protein 3 (NLRP3) inflammasome related to neuroinflammation.

Speaker: Andreas Ludwig
ADAM Family Proteases: Friends or Foes in Pulmonary Inflammation?

Several membrane-bound proteins with a single transmembrane domain are subjected to limited proteolysis at the cell surface. This cleavage leads to the release of their biologically active ectodomains, which can trigger different signalling pathways. In many cases, this ectodomain shedding is mediated by members of the a disintegrin and metalloproteinase (ADAM) family. ADAM10, in particular, can be positively and negatively involved in various physiological processes as well as in inflammatory, fibrotic and malignant pathologies. Using mice with cell specific ADAM10 deficiency we could demonstrate a role of ADAM10 in acute pulmonary inflammation. In vivo studies and vitro analysis of primary lung cells demonstrate a critical involvement of ADAM10 in cytokine production, edema formation, vascular permeability and leukocyte recruitment. Endogenous inhibitors such as TIMP1 can control excess ADAM10 activity. Such control can also be achieved by application of synthetic small molecules. In previous studies we have described the small molecule inhibitor GI 254023X (GI) which can rapidly block ADAM10 by binding to the active site. Now, we demonstrate that this inhibitor also induces long term downregulation of the protease from the cell surface associated with degradation and release of the protease in extracellular vesicles. The resulting loss of ADAM10 shedding activity is reversed by de novo synthesis of the protease. Furthermore, intraperitoneal administration of GI in mice led to profound loss of surface expressed ADAM10 on leukocytes derived from peripheral blood or different organs. Finally, GI effectively suppressed induction of acute lung inflammation. Thus, synthetic and natural inhibitors can mediate long term systemic inhibition of ADAM10 by depletion of the protease. These findings are of importance for development of optimal treatment strategies using ADAM10 inhibitors. 

 

Other targets of interest are the pituitary adenylate cyclase activating polypeptide (PACAP), with a monoclonal antibody in preclinical assays, and its PAC1 receptor, in relation to which a monoclonal antibody already exists in phase II clinical trials that may give rise to a new family of antimigranous. Transient receptor potential (TRP) channels, linked to the release of neuropeptides, constitute another hypothetical target for the control of migraine that is currently under investigation, as well as acid‐sensing ion channels (ASICs), implicated in pain perception and who are inhibited by AINE, or NOD-like receptor protein 3 (NLRP3) inflammasome related to neuroinflammation.

Speaker: Inés Roger (Oral Communication)
Role of il-11 in pulmonary fibrosis associated to pulmonary hypertension

Background
Pulmonary hypertension (PH) in idiopathic pulmonary fibrosis (IPF) portends a poor prognosis. Currently, no therapy can improve survival of patients diagnosed with this disease. IL-11 molecular pathway is over-expressed in proliferative disorders however, its role in PH- associated IPF is unknown.

Objective
The aim of this study was to evaluate the expression of IL-11 in IPF patients with or without PH. Also we hypothesized that the stimulation of pulmonary artery smooth muscle cells (PASMCs) and human pulmonary artery microvascular endothelial cells (HMVEC-L) with IL-11 induced the transformation into invasive myofibroblast.

Methods
Human pulmonary artery rings, parenchyma tissue, broncho-alveolar lavage (BAL) and serum were obtained from control subjects (n=32), IPF (n=26) and IPF with PH patients (n=22) to study the expression and predominant distribution of IL-11 and IL-11Rα. The effect of recombinant human IL-11 on pulmonary artery remodeling was evaluated in isolated PASMCs and HMVEC-L.

Results
IL-11 and IL-11Rα were over-expressed in pulmonary arteries, parenchyma, serum and BAL of IPF patients with PH and in a lesser extend in IPF patients compared with control subjects. The inmunostaining and inmunofluorescence revealed a predominant distribution of IL11 and IL-11Rα in remodeled pulmonary arteries of IPF patients and in a greater extend in IPF with PH patients and no expression in control subject. IL-11 induced morphological changes in isolated PASMCs and HMVEC-L characterized by myofibroblast phenotype.

Conclusions
IL-11 and IL-11Rα are over expressed in pulmonary arteries of IPF + PH patients contributing to pulmonary artery remodeling. Pharmacologic modulation of this route may be a promising target for the treatment of this disease.

Speaker: Paula Montero (Oral Communication)
The role of MUC1 in a bleomycin induced pulmonary fibrosis mouse model

Background
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and irreversible form of fibrotic interstitial lung disease. MUC1, a membrane-bound O-glycoprotein, is considered as oncogenic molecule by altering signaling pathways involved in cellular processes related to IPF. In previous studies we have observed an up-regulation of MUC1 and its phosphorylated forms in IPF lung tissue, as well as the in vitro involvement of its cytoplasmatic tail (MUC1-CT) in IPF characteristic cellular processes.

Objective
To analyze the implication of MUC1 in IPF by the use of a bleomycin (BLM)-induced lung fibrosis MUC1- Knock Out (KO-MUC1) mouse model.

Methods
The influence of MUC1 in pulmonary fibrosis was evaluated by the difference in survival rate, lung function and tissue remodeling between BLM-induced pulmonary fibrosis WT and KO-MUC1 mice. Masson trichrome staining, micro-CT-SPECT-PET analysis and right ventricular hypertrophy index were performed. Expression of IPF markers was analysed from lung tissue by Real Time-PCR, Western Blotting and immunohistochemistry.

Results
Mortality was improved in MUC1-KO mice. WT BLM treated animals showed increased Penh values that were significantly lower in the MUC1-KO BLM group. WT BLM treated mice showed higher collagen deposition and lesser air spaces than MUC1-KO mice. Right ventricular hypertrophy and pulmonary metabolism were increased in WT BLM treated mice. By contrast, pulmonary circulation was impaired in WT BLM group. Expression of recognized fibrotic markers and mediators were induced by BLM in lung tissue from WT mice, whilst MUC1-KO animals maintained a minimal elevation. In addition, similarly to previous results, MUC1-CT phosphorylated forms were upregulated after BLM administration.

Conclusions
MUC1-CT and its phosphorylated forms are increased in BLM-induced pulmonary fibrosis mouse model. Unlike WT mice, KO-MUC1 mice are protected against IPF, improving lung function, inflammation and fibrotic lung tissue remodeling. Therefore, pharmacologic targeting of MUC1 may be a promising option for the treatment of IPF.

SESSION 7: 
New advances in Neuropharmacology

Speaker: Ricardo Borges Jurado
Inside a Secretory Vesicle. Novel Targets for Modulating the Exocytosis of Neurotransmitters

Chromaffin granules are similar organelles to the large dense core vesicles (LDCV) present in many cell types including neurons. LDCV accumulate solutes at large concentrations (catecholamines, 1 M; ATP, 200 mM; or Ca2+, 40 mM). Solutes seem to aggregate into a condensed matrix under the acidic vesicular pH (≈5.5) to elude the osmotic lysis. The affinity of solutes for LDCV matrix is responsible for the delaying release of the release of catecholamines after granule fusion. We have accumulated experimental evidence to conclude that the manipulation of intravesicular media largely affects the LDCV cargo and produce changes in the kinetics of exocytosis.

The accumulation of solutes depends on the pH gradient across membrane vesicle that is maintained by a V-ATPase. The alkalinization of secretory vesicles slows down exocytosis and causes the leak out of catecholamines and Ca2+.

We have studied the functional contribution of chromogranins (the major intravesicular proteins) and ATP to the exocytosis by manipulation of its expression and using KO animals. Also, we have explored the consequences of the lack of the main catecholamine (adrenaline) stored in LDCV using a PNMT-KO mouse.

Several drugs are accumulated inside LDCV (hydralazine, b-blockers, some antimalarial or tricyclic antidepressants) displacing catecholamines to the cytosol. In addition, these drugs are co-released with catecholamines as false neurotransmitters.

In this presentation we will summarize all of our results obtained along the last years.

-Camacho M. et al (2008) J. Biol. Chem. 283, 22383-22389.

-Díaz-Vera, JM. et al (2012) FASEB J. 26, 430-438.

-Borges, R. Purinergic Sig. 9, 5-6.

-Kopell, WN & Westhead, EW (1982) J Biol Chem. 257, 5707-5710.

-Estévez-Herrera, J. et al (2016) PNAS. 113, E4098-4106

Supported by the Spanish Ministerio de Ciencia, Innovación y Universidades (MICIU; BFU2017-82618) and. AGS and ADLI are recipient of FPI fellowships (MICIU); PM has a contract from MICIU.

Speaker: José Guzmán González
Pamam Dendrimers Effects on Neuronal Functionality in vitro
Speaker: Ramón Sotomayor Zárate
Programming of Dopaminergic Neurons by Early Exposure to Sex Hormones: is a Vulnerability Factor for Drug Addiction?
Speaker: Izaskun Buendia Abaitua
Supplementation with Melatonin Impedes Cognitive Decline in tau-related Alzheimer Models, by Restoring the Autophagic Flux, once the Pathology is Initiated

 Lack of effective treatments for such a devastating neurodegenerative disease, like Alzheimer’s disease, makes the scientific community to work on the understanding of the mechanisms underlying the pathology and on its potential pharmacology treatment. Alterations in autophagy are increasingly being recognized in the pathogenesis of Alzheimer’s disease. Melatonin is a neurohormone whose levels are decreased with aging and, most importantly, in certain neurodegenerative diseases like Alzheimer’s disease. The lack of knowledge of how autophagy failure participates in the pathogenesis of Alzheimer’s disease and the potential relationship between the reduced levels of melatonin in this patients with this clearance mechanism, prompted us to define this study. Different Tau-related models were performed. For instance, injection of AAV-hTau/GFP viral vectors and treatment/injection with okadaic acid were used to achieve ex vivo, human brain slices and in vivo tau related models. In the in vivo studies, intracerebroventricular injection of AAV-hTau increased oxidative stress, neuroinflammation and tau hyperphosphorylation in the hippocampus 7 days after the injection, without inducing cognitive impairment; however, when animals were maintained for 28 days, cognitive decline was apparent. Interestingly, late melatonin treatment, starting once the alterations mentioned above were established (from day 7 to day 28) reduced oxidative stress, neuroinflammation, tau hyperphosphorylation and caspase-3 activation; these observations correlated with restoration of the autophagy flux and memory improvement. This study highlights the importance of autophagic dysregulation in tauopathy and how melatonin treatment in the prodromal phases of the disease can restore the autophagy flux, and thereby, prevent cognitive decline. We therefore propose the development of drugs that improve the autophagy flux for the treatment of proteinopathies like Alzheimer’s disease.

PLENARY LECTURE: 
Somatic Mutations and Clonal Hematopoiesis as Drivers of Inflammation and Cardiovascular Disease

Speaker: José Javier Fuster
Somatic Mutations and Clonal Hematopoiesis as Drivers of Inflammation and Cardiovascular Disease

Recent exome sequencing studies in humans have shown that normal aging is unavoidably associated with the accumulation of somatic mutations in the hematopoietic system, which in some cases provide a competitive growth advantage to the mutant cell and allow its clonal expansion. Remarkably, this somatic mutation-driven clonal hematopoiesis has been associated with increased mortality due to both cancer and non-cancerous conditions, most frequently cardiovascular disease. This talk will summarize the results of several epidemiological and experimental studies supporting the possibility that clonal hematopoiesis represents a new and unexpected risk factor and driver of atherosclerotic cardiovascular disease and, potentially, many other age-related inflammatory conditions. Knowledge gained in these studies could eventually lead to the design of personalized therapies or preventive care strategies for individuals carrying specific somatic mutations in blood cells.

SESSION 8: 
Free Radicals in traslational Pharmacology

Speaker: María José Alcaraz
Relevance of Nrf2 and Heme Oxygenase-1 in Articular Disease

A wide range of evidence indicates that Nrf2 is able to control oxidative stress and inflammation as well as innate and adaptive immune responses. Nrf2 inhibits the production of reactive oxygen species and mediators driving chronic inflammation and tissue destruction. This transcription factor can decrease differentiation, proliferation and activity of antigen-presenting cells, T cells and B cells while enhancing these processes in regulatory T cells. The appropriate balance of Nrf2 is necessary for cartilage metabolism. In addition, Nrf2 plays a key role in the maintenance of bone microarchitecture. Heme oxygenase-1 (HO-1) induction is a consequence of Nrf2 activation. Therefore, oxidative stress and pro-inflammatory cytokines upregulate HO-1 whereas anti-inflammatory cytokines have the opposite effect. HO-1 upregulation or administration of its metabolites results in anti-inflammatory and antioxidant effects in articular diseases and it may protect against cartilage destruction and inflammatory bone loss. HO-1 decreases the production of oxidative stress, pro-inflammatory agents and catabolic enzymes and controls the dysbalance between anabolic and catabolic processes in articular cells. Both Nrf2 and HO-1 can be targets in the control of joint degradation and bone alterations. There is a need for improved strategies to regulate their activity which may open up new therapeutic opportunities for the treatment of joint conditions.

Speaker: Ana Casas
Free Radicals in translational Pharmacology: NOS, NOX & sGC as a network pharmacology-based approach for brain ischemia

Existing drugs fail to provide benefit for most patients. The efficacy of drug discovery is in a constant decline. This poor translational success of biomedical research is due to false incentives, lack of quality/reproducibility and publication bias. In fact, drug discovery faces an efficacy crisis to which ineffective mainly single-target and symptom-based rather than mechanistic approaches have contributed. Systems medicine opened a new concept of therapeutic treatment focused on the so-called network pharmacology, where several targets modulated at the same time lead to the first effective therapy for high unmet medical need indications with no treatment so far. 

In stroke, we therefore validated 3 single ROS-related enzymes, i.e. NADPH oxidase, NO Synthase and soluble Guanylate Ciclase as promising therapeutic targets for brain ischemia within a network-pharmacology strategy. Pharmacological modulation of these targets leads to less infarct size, reduced blood-brain barrier leakage, improved neuro-motor functioning and direct neuroprotection. Our therapeutic approach is now in the last pre-clinical step (large animal validation) towards a clinical trial, currently in design. 

Speaker: Pietro Ghezzi
The Oxidative Stress Theory of Disease

The formulation of theories about mechanisms of disease has led to major advancements in medicine. These include, for instance, the germ theory of disease and the cytokine theory of disease. The oxidative stress (OS) theory of disease implicates that OS is a causal factor in some diseases and that antioxidant molecules, by scavenging reactive oxygen species (ROS) could represent a therapeutic approach. However, although it was first hypothesized in 1956 with the “free radical theory of ageing”, so far there are no antioxidants that have been approved as medicinal products by regulatory agencies. Despite this, and the fact that OS has been implicated practically in every disease, there is a multibillion dollar market of antioxidant supplements. Of course new research will identify new antioxidant molecules and those may be found effective, but there is a possibility that there is an intrinsic weakness in the OS theory of disease.

We will analyze the weakness of this theory highlighting various problems encountered when analyzing causation in biology. These include the epistemological problem of drawing a causal link, the lack of specific experimental tools, the lack of a way of grading experimental evidence that fits the criteria of evidence-based medicine, and the problem of extrapolating conclusions from well-defined experimental model to the clinical situation.

In particular, we will focus on the difficulty of measuring ROS, which forces us to use surrogate biomarkers. Finally, we will discuss the role of OS in diseases with multiple causes and how this impact on the interpretation of results from statistical analysis using significance testing.

References: (1) Ghezzi P, Davies K, Delaney A, Floridi L. Theory of signs and statistical approach to big data in assessing the relevance of clinical biomarkers of inflammation and oxidative stress. Proc Natl Acad Sci U S A. 2018;115:2473-2477.  (2) Ghezzi P, et al. Oxidative Stress and Inflammation Induced by  Environmental and Psychological Stressors: A Biomarker Perspective. Antioxid Redox Signal. 2018;28:852-872. (3) Ghezzi P, Jaquet V, Marcucci F, Schmidt HHHW. The oxidative stress theory of disease: levels of evidence and epistemological aspects. Br J Pharmacol. 2017;174:1784-1796.

Speaker: Cristina Estornut (Oral Communication)
Evaluation of Nrf2 Activators for the Treatment of Chronic Obstructive Pulmonary Disease (COPD)

Background
Chronic Obstructive Pulmonary Disease (COPD) is a progressive inflammatory lung disease primarily caused by chronic exposure to cigarette smoke. Oxidative stress is one of the principal mechanisms involved in the physiopathology of the disease. Nuclear Factor Erythroid 2-related (Nrf2) is critical in protection against oxidative stress by inducing expression of antioxidant enzymes and a significant decrease in its expression has been observed in COPD patients.


Objective
The aim of this study was to characterize the effects of Bardoxolone, Omavexolone and Obacunone as antioxidant drugs in COPD.


Methods
Peripheral blood neutrophils from COPD and healthy volunteers and Primary Human Bronchial Epithelial cells were pre-incubated with the cited drugs at different concentrations and stimulated with cigarette smoke extract (CSE). Expression of antioxidant genes, cytokine release, GSH levels and apoptosis were measured by RT-PCR, ELISA, luminescent assay and flow cytometry, respectively.


Results
Expression assays using lung tissue and neutrophils from COPD and healthy volunteers showed a negative correlation between the expression of antioxidant genes and the severity of the disease. After stimulation with CSE and drug treatment, cells displayed an increase in expression of antioxidant genes, as well as, an inhibition in the release of inflammatory cytokines. In addition, GSH assays showed that Bardoxolone, Omavexolone and Obacunone were able to activate Nrf2 with EC50 values of 6.4nM, 15.3nM and 38.7μM, respectively. Moreover, these drugs were highly effective in apoptosis inhibition.


Conclusions
Bardoxolone, Omavexolone and Obacunone show a huge antioxidant response against CSE-induced COPD by Nrf2 activation. Thus, these drugs may represent a promising therapeutic option in COPD.

Speaker: Miguel Tofiño-Vian (Oral Communication)
Adipose tissue mesenchymal stem cell-derived extracellular vesicles as a biological therapy in osteoarthritic cells

INTRODUCTION: Osteoarthritis (OA) is a joint condition associated with articular cartilage loss, low-grade synovitis and alterations in subchondral bone and periarticular tissues. In the last years, the possible therapeutic application in OA of adipose tissue-derived mesenchymal stem cell (ASC)-extracellular vesicles (EVs) has aroused considerable interest among the scientific community. In this work, we have integrally assessed the immunomodulatory and antisenescent dose-dependent properties of ASC microvesicles (MV) and exosomes (EX) in human chondrocytes (OAC) and osteoblasts (OB) from OA patients.


METHODS: ASC were isolated from fat of patients who undergone abdominoplasty, and were cultured with appropriate media supplemented with EV-free serum. EV were isolated from AD-MSC conditioned medium by differential centrifugation and characterized by resistive pulse sensing, mass spectrometry and electron microscopy. OAC and OB were isolated from knee specimens of advanced osteoarthritis patients, stimulated with IL-1β (10 ng/mL) and treated with MV (3.6×107 particles/mL) or EX (7.2×107 particles/mL) for 24h. Then, inflammatory, oxidative and senescence markers were measured by several methods such as ELISA, radio-immune assay, fluorometry and confocal microscopy. Data was analysed by ANOVA followed by Dunnett’s test.
RESULTS: Both in OAC and OB, EVs were able to downregulate the levels of IL-6, TNFα, PGE2, MMP activity and NO production, and stimulated the production of the anti-inflammatory IL-10. In OAC, EVs stimulated the production of collagen II, an effect tied to the presence of annexin-A1 in MVs. In OBs, EVs reduced several senescence makers such as lipid peroxidation, inner mitochondrial layer potential and β-galactosidase activity.


CONCLUSION: Administration of EV may have potential pharmacological applications to control inflammatory processes, extracellular matrix degradation and intracellular remodelling in osteoarthritic cells. In this regard, we propose ASC-derived EVs a putative biological therapy for chronic inflammatory conditions such as OA.

SESSION 9: 
Poster Presentation (8 selected posters)

Speaker: Elena Domingo
CCL11/CCR3 axis is involved atherosclerosis development

Introduction: Primary hypercholesterolemia (PH) is a lipid disorder characterized by elevated levels of cholesterol and low-density lipoprotein (LDL). Generally, deleterious environmental factors such as hypercholesterolemic diets and obesity are linked to the disease. A low-grade systemic inflammation is associated with PH, which might explain the higher incidence of cardiovascular diseases such as atherosclerosis, one of the leading causes of morbidity and mortality in Western countries. The early atherosclerotic lesion involves an inflammatory response, the intimal accumulation of T lymphocytes and lipid-laden macrophages, continuing throughout the atherogenic process. Eotaxin-1/CCL11 has been detected in human and mouse atherosclerotic aortas; however, its role in the atherosclerotic lesion development remains elusive.

Objective: To evaluate the impact of eotaxin-1 receptor (CCR3) in PH patients and in the lesion formation in apoE−/−CCR3+/+ mice versus apoE−/−CCR3−/− mice.

Materials and Methods: Whole blood from 22 PH patients, 21 age-matched controls and two-month-old apoE−/−CCR3+/+ or apoE−/−CCR3−/− mice subjected or not to an atherogenic diet (10.8% fat, 0.75% cholesterol) during two months, was analysed by flow cytometry to determine CCR3-expression in different leukocyte subsets. Circulating chemokines were determined by ELISA. Lesion formation, inflammatory infiltration, collagen, necrotic core, vascular smooth muscle cells, mast cells, eosinophils and eotaxin-1 content were determined through histological and immunohistochemical techniques. Results: PH patients and atherogenic diet-fed apoE−/−CCR3+/+ mice show elevated percentage of CCR3-expressing leukocytes. Atherogenic diet-fed apoE−/−CCR3−/− mice showed increased lesion formation, collagen content and macrophage, T lymphocyte and mast cells infiltration respect to apoE−/−CCR3+/+. Lesion eotaxin-1 expression of atherogenic-fed apoE−/−CCR3+/+ mice was much higher than that detected in apoE−/−CCR3−/−.

Conclusion: CCL11/CCR3 axis may exert a protective effect during the development of atherosclerotic processes. Funding: This work was supported by the Spanish Ministry of Economy and Competiveness [grant numbers SAF2014-57845-R, SAF2017-89714-R]; Carlos III Health Institute and the European Regional Development Fund [grant numbers PI15/00082, PIE15/00013, PI18/00209].

Speaker: Álvaro San Hipólito-Luengo
The adipokine visfatin induces endothelial dysfunction through TLR-4and NLRP3 inflammasome activation.

 Visfatin is a multi-faceted adipokine whose expression and secretion are enhanced in obesity. It has been suggested that visfatin can promote vascular inflammation and endothelial dysfunction through its enzymatic Nampt activity.

Speaker: Sergio Montserrat De La Paz
Characterization and evaluation of hemp protein hydrolysates on neuroprotection

Hemp (Cannabis sativa L.) seeds are well known for their potential use as a source of food-nutrients, dietary fiber, and bioactive compounds. A hemp seed protein isolate (HPI), prepared from defatted hemp seeds, was hydrolyzed by Alcalase or Alcalase-flaoryzime under specific conditions. The resulting hydrolysates were evaluated to the selection of potentially bioactive hemp seed protein hydrolysates (HPHs) through DPPH scavenging and angiotensin-converting enzyme-inhibitory activities. In vitro cell-free experiments led to the identification of two 2 bioactive HPHs, HPH3 and HPH7, which were used at 50 and 100 μg/μL on BV2 microglial cells in order to evaluate the anti-inflammatory and neuroprotective activities. RT-qPCR techniques were used to analyse the mRNA transcriptional levels. Our results showed that HPH3 and HPH7 down-regulated the mRNA transcriptional levels of IL-1β, and TNF-α in BV2 microglial cells stimulated with lipopolysaccharide. In addition, HPH3 and HPH7 promote M2 microglia polarization, down-regulating iNOS and up-regulating Arg-1. This study suggests for the first time that HPHs consumption may improve neuroinflammatory and chronic inflammatory states, confirming that hemp seeds are a valuable source of bioactive compounds.

Speaker: María Soledad Avendaño
Obesity-induced hypogonadism: Pathophysiological roles of a novel hypothalamic miRNA/Kisspeptin pathway and potential therapeutic implications

Obesity is a life threatening condition associated with numerous comorbidities. Among them, central hypogonadism, i.e., low circulating levels of testosterone, has been recently suggested as a putative contributor to the metabolic complications of obesity, especially in men. Yet, the mechanisms for obesity-induced hypogonadism (OIH), and its actual roles in the generation/evolution of the metabolic alterations of obesity remain ill defined. Yet, recent data has suggested that OIH may be caused by suppression of hypothalamic Kiss1 system; kisspeptins being potent activators of the reproductive axis that ultimately stimulate testosterone secretion. However, the mechanisms for Kiss1 suppression in obesity remains unknown.
Our recent evidence suggests that microRNAs (miRNAs) are putative regulators of the Kiss1 system. In this work, we aimed to identify novel miRNAs capable of regulating kisspeptin expression and to evaluate their potential contribution to OIH. Bioinformatic analyses on KISS1 gene were conducted with different algorithms (http://www.targetscan.org/; http://www.ebi.Ac.uk/enright-srv/microcosm /htdocs/targets/v5/; http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/; http://www.microrna. org/microrna/home.do) to seek for potential miRNA regulators. Selection of miRNA candidate(s) was based on the following criteria: 1) to be identified in at least two different databases; 2) to show an evolutionary conserved seed region (rat, mouse, human); and 3) to be deregulated by metabolic alterations, associated with changes in hypothalamic kisspeptin expression. Using these criteria, miR-A (anonymized for reasons of ongoing patent protection) was selected as a robust putative modulator of KISS1. This was tested biochemically using a luciferase reporter assay, which documented a repressive interaction of miR-A at the 3’UTR of KISS1. Further confirmation was obtained in vivo, using a Target Site Blocker (TSB) approach. TSB, tailored to block the repressive interaction of miR-A selectively at Kiss1 3’UTR, kisspentin-10 and/or testosterone (T) were administered to a male rat model of OIH, with severe suppression of T and gonadotropin (LH) levels, and marked metabolic alterations: increased systolic blood pressure (SBP), glucose intolerance and insulin resistance. TSB administration not only restored T and LH levels and increased hypothalamic kisspeptin, but also ameliorated insulin resistance, glucose intolerance, SBP and cardiac hypertrophy, without detectable changes in absolute body weight. In contrast, treatments with kisspeptin-10 or T failed to restore the alterations induced by OIH. Our results are the first to provide conclusive evidence about the relevant role (and eventual therapeutic value) of a novel central miRNA/ kisspeptin pathway in OIH, and strongly suggest that central hypogonadism is a major contributor for the metabolic complications of obesity.

Speaker: Magdalena Nikolaeva Koleva
In vitro characterization of a TRPV1 soft antagonist

TRPV1, a member of the transient receptor potential (TRP) family, is a nonselective calcium permeable ion channel gated by diverse stimuli (capsaicin, T, pH and voltage). TRPV1 plays a fundamental role in the pathogenesis of dermatological diseases specially related to inflammation, pain and pruritus. Accordingly, the development of new TRPV1 antagonists is yet a high priority. To address this issue, we have designed capsaicinoid molecules that incorporate an ester group in the lipophilic moiety. These compounds have the advantage of undergoing deactivation (soft drug concept) once their desired local effect has been exerted, thus preventing its long-term dermal accumulation. Here, we report the pharmacological evaluation of AG1529 on heterologously expressed human TRPV1 by patch clamp using whole cell configuration. Electrophysiological characterization of AG1529 was further analysed on primary sensory neuronal networks established onto planar multielectrode arrays (MEA). We found a mean IC50 value of 0.92 (0.28 to 3.01) µM. AG1529 reversibly blocked the channels, with completed washout after 5 min of stopping its instillation. Furthermore, AG1529 is a capsaicin competitive antagonist as it displaced the agonist EC50 to higher values. Molecular modelling substantiates an interaction with the capsaicin binding site in TRPV1. AG1529 also blocked other modalities of TRPV1 activation, and had a marginal inhibitory activity of TRPA1 and TRPM8 as compared with that exerted on TRPV1. MEA measurements show that AG1529 attenuated capsaicin induced neuronal firing in both basal and algesically sensitized neuronal cultures. These results unveil the mode of action of AG1529, and substantiate the tenet that this compound is a promising lead for skin therapeutics.

Speaker: Andrea García Silva
Quantification of functional bias of serotonin 5-HT2A receptor agonists and validation of structural hypotheses on functional selectivity at 5-HT2A receptor

5-HT2A receptor (5-HT2AR) was one of the first G protein-coupled receptors (GPCRs) for which functional selectivity was described.1 A recent computational study proposed agonist interaction with residue S242 (S5.46) of 5-HT2AR as relevant for ligand bias towards the inositol phosphate (IP) signalling pathway modulated by the receptor, whereas interaction with residue N343 (N6.55) would favour bias towards the arachidonic acid (AA) pathway.2 These observations allowed to select the serotonin (5-HT) derivatives 5-nitro-1H-indole-3-ethanamine (NitroI), silent at the AA pathway, and 3-(aminoethyl)1-methylindol-5-ol (MetI), which preferentially activated the AA pathway. Here we aimed at quantitatively determining the functional bias of these agonists as well as at directly testing by site-directed mutagenesis the structural hypotheses formulated on functional selectivity at 5-HT2AR.
IP and AA signalling of 5-HT, NitroI and MetI was evaluated at 5-HT2A-WT, 5-HT2A-S242A and 5-HT2A-N343A receptors. Functional bias was quantified applying the operational model of functional selectivity.
The point-mutations did not compromise the affinity of [3H]-Ketanserin for 5-HT2AR. At 5-HT2A-WT, MetI showed bias towards the AA pathway with ΔΔlog (τ/KA) (IC 95%) = 1.27 (0.73 – 1.82), in agreement with the prediction for this derivative. This bias diminished at 5-HT2A-S242A (ΔΔlog (τ/KA) (IC 95%) = 0.55 (-0.16 – 1.27)), consistent with residue S242 as relevant for IP bias. However, the improvement in affinity and potency of MetI specifically at the IP pathway supports changes in its binding mode in this mutant, with other interactions contributing to IP signalling. The N343A mutation preserved AA signalling of 5-HT and MetI as well as MetI bias towards AA (ΔΔlog (τ/KA) (IC 95%) = 1.59 (0.63 – 2.56)), whereas unexpectedly reduced the IP potency of both agonists. Even considering possible limitations of our experimental approach, the results suggest that other agonist properties indirectly related to the interaction patterns investigated may contribute to the bias of compounds NitroI and MetI.

 

Speaker: Beatriz Ballester
Muc1-ct as an ipf potential druggable target

Background
MUC1 overexpression is one of the most common alterations in human cancers. This overexpression is associated with accumulation of MUC1-C in the cytoplasm and targeting of MUC1-C to the nucleus by MUC1-C oligomerization by a CQC motif in MUC1-cytoplasmic tail (CT). In previous studies we showed that MUC1 is also overexpressed in Idiopathic pulmonary fibrosis (IPF) lungs, and play an essential role in the cellular transformations and processes related to IPF. However, the participation of MUC1-CT signalling in IPF is currently unknown.

Objective
To analyze the implication of MUC1-CT in IPF

Methods
The intracellular mechanism of MUC1 in IPF was evaluated by immunoprecipitation and immunofluorescence studies in IPF lung tissue and alveolar type II cells and lung fibroblasts stimulated with TGFβ1, and by the use of the peptide GO-201 (an inhibitor of MUC1-CT CQC motif that attenuates targeting of MUC1-C to the nucleus) in these cells. This inhibitor was used to study the effect of MUC1-CT on epithelial to mesenchymal (EMT) and fibroblat to myofibroblasts (FMT) transitions, cellular senescence and cellular proliferation. Attenuation of lung fibrosis development in a GO-201 treated bleomycin-induced pulmonary fibrosis mouse model was also assessed.

Results
Immunoprecipitation and immunofluorescence studies showed that under fibrotic conditions MUC1-CT forms a complex with p-Smad2/3 and active-β-catenin, translocating into the nucleus to activate fibrotic genes in human cells and human lung tissue. Furthermore, inhibition of nuclear MUC1-CT translocation by GO-201 indicated that MUC1-CT activation and translocation into the nucleus is essential to induce EMT and FMT cellular transformations, as well as, ATII and fibroblast cell senescence and fibroblast proliferation. Finally, lung tissue remodeling was attenuated in GO-201 treated mice.

Conclusions
MUC1-CT is essential to induce IPF progression. Therefore, pharmacologic targeting of MUC1-CT may be a promising option for the treatment of IPF.

Speaker: María Extremera Mazuela
Mechanism of resistance to EGFR-Targeted therapy induced by nicotine in human lung cancer

Cigarette smoking is not only correlated with the onset and progression of a variety of human tumors, including lung cancer, but continued smoking after cancer diagnosis is known to reduce the effectiveness of conventional chemotherapy and EGFR-targeted therapy. Certain nicotinic acetylcholine receptor (nAChR) subtypes expressed in lung tumor cells play an essential role in the acquisition of the above resistance to anti-tumor drugs after their activation by certain tobacco-specific constituents, like nicotine. Although the mechanism implicated in the nicotine-induced resistance to conventional chemotherapy has been widely investigated, the mechanism that underlies resistance to EGFR-targeted therapy remains unexplored. Here, by using a combination of molecular biology, biochemical, pharmacological and flow cytometry techniques, we evaluate the nicotine effect on cell viability, apoptosis, cell cycle distribution and gene expression levels of several nAChR subunits in the human lung adenocarcinoma cell line HCC827 (EGFR mutated) exposed to erlotinib or osimertinib (72 h). Our results show that nicotine reduces the cytotoxicity of erlotinib (IC50 = 0.153 ± 0.01 μM vs. 1.93 ± 0.26 μM; p ≤ 0.05) and osimertinib (IC50 = 3.87 ± 0.10 nM vs. 20.06 ± 0.10 nM; p ≤ 0.05) through the inhibition of their apoptotic effects. Moreover, nicotine partially prevents the erlotinib-mediated reduction of the cell fraction in the S phase. Both drugs up-regulate the expression of most of the nAChR subunits tested, including the α7, leading to an increase in the number of nicotine binding sites in the tumor cells. Taken together, our results identify various mechanisms by which nicotine causes tobacco-mediated resistance to EGFR-targeted therapy in lung cancer. Although further studies will be required to elucidate the nAChR subtype responsible of the above nicotine effects, our findings may be clinically relevant and increase our understanding of the mechanisms involved in tobacco-mediated resistance to new cancer-targeted therapy.

SESSION 10:
Aging from mechanisms to pharmacological perspectives

Speaker: Jose Viña
Aging from Mechanisms to Pharmacological Perspectives

Understanding the mechanisms of aging is of primary importance to promote longevity, but also –and critically important- healthy aging.

The concepts of aging, longevity and frailty will be discussed (1). Based on considerable information on the mechanisms of aging gathered in the last twenty years, we can provide basis for the promotion of longevity, but also of healthy aging, a concept closely related to the more clinically accepted idea of frailty, a geriatric syndrome that leads to disability and death.

Healthy aging can be promoted by interventions like programmed exercise, that we consider as a drug (2), and one specially useful for the elderly (3),

Nutritional interventions will also be discussed with special emphasis on the importance of protein.  Finally the role of conventional pharmacological interventions – with emphasis on drugs like metformin or antioxidants- will be emphasized.

The main  corollary that will be discussed , i.e. that interventions to treat frailty and delay disability, are of the utmost importance for individual health but also for society will serve to close the presentation (4)

1.- Hayflick, L.-  Biological Aging Is No Longer an Unsolved Problem Ann. N.Y. Acad. Sci 1100, 1- 13 (2007)

2.- Vina J et al. Exercise acts as a drug.  Pharmacological benefits of exercise  British Journal of Pharmacology  167:1-12 (2012)

3.- Vina J et al.  Pharmacological properties of physical exercise in the elderly, Current Pharmaceutical Design, 20, (2014)

4.- Nascimento C et al Sarcopenia, frailty and their prevention by exercise Free Rad Biol Med  132, 42- 49 (2019) 

 

Speaker: Jorge Fuentealba Arcos
P2xr Overexpression contribute to Beta Amyloid Toxicity: New Pharmacological Target to AD

Soluble oligomers of amyloid beta peptide (SOAβ) have been considered as central factors in Alzheimer’s disease (AD). Aβ peptide is generated through the sequential cleavage of the amyloid precursor protein (APP), a process that requires the previous endocytosis of APP and that can be modulated by the multidomain adaptor protein Fe65. This protein is able to regulate the transcription of key genes directly related to AD pathogenesis, encoding proteins like APP and BACE 1. On the other hand, we have described that chronic SOAβ treatment induces an increase in the expression of the P2X2 purinergic receptor in PC12 cells and hippocampal neurons. Additionally, it has been described that the P2X2a isoform has an intracellular domain that can interact with Fe65, a segment which is absent on the P2X2b isoform. We found that SOAβ treated cells displayed an increase in evoked ATP currents (C: 100 ± 50%; SOAβ: 231 ± 70%; n=9). Additionally, immunocytochemistry (ICC) experiments demonstrated that these cells exhibited an increase in their P2X2R immunoreactivity (C: 100 ± 1 %; SOAβ: 149 ± 15%; n=5). Moreover, cells treated chronically with SOAβ showed a reduction in the Fe65 nuclear-cytoplasmic (N-C) ratio (C: 100 ± 6%; SOAβ: 80 ± 4%; n=5). A similar behavior was observed in PC12 cells transfected to express the P2X2a isoform, but not in those transfected with P2X2b (C: 100 ± 5%; P2X2a: 70 ± 6%; P2X2b: 95 ± 6%; n=3). Colocalization analyses demonstrated that SOAβ decreased the colocalization of Fe65 with APP (C: 100 ± 17%; SOAβ: 47 ± 12%; n=5); results that correlate with the increase observed in the colocalization of APP with clathrin (C: 100 ± 8%; SOAβ: 127 ± 8%; n=4) and Rab5 (C: 100 ± 6%; SOAβ: 132 ± 16%; n=5). In conclusion, these results suggest that chronic SOAβ treatment promotes the endocytosis of APP, potentiating its amyloidogenic processing. Additionally, the calcium dyshomeostasis/overload induced by P2X2R overexpression, alter the activation and localization of CAMKIIα, in the context of AD. Using molecular biology techniques, we observed that after chronic SO-Aβ treatments, mice hippocampal neurons showed an increase on the levels of P2X2R compared to the control cells (C: 100.0 ± 6.4%; SOAβ: 130.1 ± 10.7%, n=5). This was correlated with increased Ca2+ signal evoked by ATP (C: 100.0 ± 12%, SOAβ: 194 ± 24%, n=4). Immunocytochemistry approaches on mice hippocampal neurons, showed that the overexpression of P2X2R induced changes on the immunorreactivity pattern of pCAMKIIα (in soma and neurites), which induced alterations on the cells morphology, and electrophysiological recordings assessed by Sholl Analysis and Patch Clamp, respectively. These results suggest that P2X2R overexpression can potentiate the toxicity of SO-Aβ, due to the chronic Ca2+ overload and inactivation of CAMKIIα, and thus, altering the mechanisms of neuronal plasticity, the basis of the pathophysiological mechanism of AD.

Speaker: Guillermo Díaz
Cardiac Fibroblast Role on Inflammatory Process: Interaction with Immune Cells

The Doctoral Programme in Pharmacology was established in the year 2002, with the participation of the Faculties of Chemical & Pharmaceutical Sciences, Medicine, Sciences and the Institute of Nutrition & Food Technology (INTA). From 2003, had received a national accreditation by the National Accreditation Institute (CNA-Chile).

Main features of the Doctoral Programme in Pharmacology

The programme entails a face-to-face, full dedication schedule. Candidates hold undergraduate degrees in chemistry, biochemistry, biology and pharmaceutical sciences, including medicine, dentistry, veterinary medicine and biotechnology. Since 2009, the candidate postulates by an on-line application system (https://postulacionpostgrado.uchile.cl). Successful applicants can apply to a CONICYT Scholarship or to any other National or International Granting Institutions, including support by the Faculty of Chemical & Pharmaceutical Sciences, University of Chile. Formal activities are based on two mandatory courses: Advanced Molecular Pharmacology, and Experimental Pharmacology, which focus on molecular modelling, genomics, pharmacological chemistry and clinical pharmacology. It is mandatory that the doctoral thesis work, must generate at least one article published by an international ISI-indexed Journal, where the candidate as a first author. All graduates from the Program are inserted in Research and Academic Institutions, both in the country or abroad. Several graduates proceed to postdoctoral positions in internationally recognised institutions, either locally, or in Europe and North America. The insertion rate is 100% for the total graduate cohorts.

Academic core

At present, the academic core is composed of 34 faculty members with the highest national and international prestige, selected among associate and full professors from the University of Chile. Based on the expertise of academic Core, six lines of research have been established, which warrant proper competitiveness, i.e. Cardiovascular, Cancer, CNS, Immunology, Antimicrobial and Chemotherapy, and Drug Development. Core have collectively authored a total of about 700 ISI articles in the past last 10 years. The Core has also files and obtained 16 patents reflecting an outstanding record.

Achievements

From 2002, more than 90 PhDs in Pharmacology have graduated from the Program. In the past few years, an average of 10 candidates per year have been accepted in the Program, and at present there are 47 candidates carrying out their thesis work. The demand for admission has also increased substantially, in particular by students from other Latin American countries (i.e. Colombia, Venezuela, Costa Rica, Ecuador, Cuba, Peru), thus presenting the Program as attractive choice in the region. The Doctoral Programme in Pharmacology has substantially prioritized international networking, including the University of Regensburg, Germany, for the development of epigenetics; and the University of Mississippi, USA, for Clinical Pharmacology. There are several agreements in progress with the Universidad Autónoma de Madrid, Spain, focusing on system pharmacology, and UNICESUMAR, Paraná, Brazil, focusing on veterinary.

Speaker: Mercè Pallás (Oral Communication)
Targeting soluble epoxide hydrolase to improve neurodegenerative traits in SAMP8 mouse

Aging is the most important risk factor of neurodegenerative disorders and is associated with cognitive impairment and dementia. Recent studies demonstrate that inflammation is a key event of the transition of normal aging to progressive neurodegeneration. Soluble epoxide hydrolase (sEH) is a key enzyme in the arachidonic acid (AA) cascade. Epoxyeicosatrienoics acids (ETTs) demonstrated anti-inflammatory among others biological effects. sEH hydrolyses ETTs to dihydroxyeicosatrienoic acids (DHETs) that loss beneficial effects. Our interest was to test a new family of sEH inhibitors (sEHi), UB-EV-52, with a new chemical structure, comparing with TPPU and AS-2586114, a well known sEHi, in a murine model of cognitive decline associated with aging, the senescence accelerated mice prone 8 (SAMP8).

Neuroprotective effects of 5mpk UB-EV-52, AS-2586114 and TPPU in 5-months-old SAMP8 were evaluated. Drugs were administered in drinking water for 4 weeks. UB-EV-52, TPPU and donepezil treated SAMP8 improved cognitive impairment (novel object recognition test) compared to SAMP8 control (SAMP8-Ct) mice group. Likewise, a reduction in neuropathological markers such as APP, sAPPα and –β, ADAM10 and BACE1, sAPP levels and abnormal tau hyperphosphorylation (Ser396 and Ser404) by Western blot were determined in treated SAMP8. Furthermore, changes in gene expression of inflammatory and oxidative stress markers such as Il-6, Cxcl10, Ccl3 and iNOS, Aox1 and Aldh2 were found in SAMP8 mice treated with sEHi compared to SAMP8-Ct. In addition, analysis of cytokine levels of IL-1β and TNF-α and hydrogen peroxide levels by ELISA showed a significant reduction in treated SAMP8 mice. Finally, a cellular thermal shift assay (CETSA) was performed to verify ex vivo target engagement of UB-EV-52 and TPPU in SAMP8 brain.

In conclusion, we demonstrated that sEHi might be an effective treatment for neurodegenerative disorders associated with aging leading to an improved cognitive impairment, reducing the neuroinflammation and oxidative stress.

Financial support from Ministerio de Economía y Competitividad (Projects SAF2016-33307 and SAF2017-82771) is acknowledged. S. C., R. L. and D.P-I. thank the Universitat de Barcelona (APIF grant), Ministerio de Educación, Cultura y Deporte (FPU program) and the IBUB, respectively, for PhD Grants.

Speaker: Alejandra Romero Martínez (Oral Communication)
The angiotensin-(1-7)/Mas receptor axis attenuates human endothelial cells senescence through the activation of klotho and Nrf2.

Background and aims: Endothelial cell senescence, is one of the major mechanisms contributing to vascular aging. It is characterized by cell growth arrest and the acquisition of a senescence-associated secretory phenotype (SASP), which predisposes to vascular disease. Here, we explored the capacity of Angiotensin (Ang)-(1-7), a member of renin-angiotensin system (RAS), to counteract human umbilical vein endothelial cell (HUVEC) senescence triggered by RAS-dependent and – independent stressors, such as Angiotensin (Ang) II or interleukin (IL)-1β. Besides, we aimed to identify intracellular pathways mediating its potential protective actions.

Methods: Cultured HUVEC were stimulated with Ang II (100nM) or IL-1β (2,5 ng/ml) for 18h. Endothelial cell senescence was assessed by positive senescence-associated β-galactosidase staining (SA-β-gal+) and by total and telomeric DNA damage. Protein levels, leukocyte adhesion to HUVEC monolayers and IL-6 secretion were determined by Western blot, flow chamber assays and ELISA, respectively.


Results: Both Ang II (100 nM) and IL-1β enhanced the fraction of SA-β-gal+ cells, the expression of ICAM-1 and VCAM-1 adhesion molecules, leukocyte adhesion and IL-6 secretion, used as markers of the SASP. Ang-(1-7) (100 nM) counteracted the pro-senescence action elicited by both Ang II and IL-1β through a mechanism that was blocked by the Mas receptor antagonist, A779 (1 µM). Ang-(1-7) also increased endothelial klotho levels, while klotho silencing blunted the anti-senescence capacity of the heptapeptide. Both Ang-(1-7) and recombinant klotho (1 nM) activated the cytoprotective Nrf2/heme oxygenase-1 (HO-1) pathway. HO-1 inhibitor tin protoporphyrin IX (1 μM) prevented the anti-senescence evoked by Ang-(1-7) or recombinant klotho.


Conclusions: The present study identifies Ang-(1-7) as a tool to protect agains human endothelial cells senescence through the consecutive activation of klotho and the Nrf2/HO-1 axis. Developing new Ang-(1-7)/Mas axis activators or mimetic drugs may prove useful to counteract endothelial cell senescence and premature vascular aging.

SESSION 11:
New Advances in the Pharmacology of Cancer

Speaker: Patricia Rijo
Abietane diterpenoids from Plectranthus spp. as Lead Protein Kinase C Modulators for Cancer Therapy

Cancer is one of the highest causes of death worldwide. Protein kinase C (PKC) is a family of serine/threonine kinases divided into three groups according to their regulatory domain structure and cofactors requirement for activation: classical, novel, and atypical PKCs. Recently, PKC family isoforms have been the focus of intense research, and recognized as therapeutic targets in anticancer drug development.  [1]. Diterpenoids are commonly found in the Plectranthus spp., and have a widespread spectrum of biological activity, namely anticancer properties [2]. The diterpenoid 7α-acetoxy-6β-hydroxyroyleanone (AHR) isolated from P. grandidentatus displays low cytotoxicity and the basic requirements approaches for the development of pharmaceutical formulations based on AHR as a lead. These AHR features includes an extraction optimization and structural and thermal properties characterization [3]. These features suggests that AHR can be used as a lead for drug development. Considering this, a small library of abietane derivatives was tested for their ability to activate PKC isoforms from classical (alpha, α; beta, β), novel (delta, d; epsilon, e) and atypical (zeta, z) subfamilies, using a previously developed yeast-based screening assay to search for modulators of PKC isoforms [4]. The results obtained revealed potent activators of PKC family proteins, namely: a selective activator of PKCd, the 7α-acetoxy-6β-benzoyloxy-12-O-benzoylroyleanone (Roy-Bz). The patented diterpenoid RoyBz was prepared using AHR as starting material. Roy-Bz potently inhibited the proliferation of colon cancer cells by inducing a PKCd-dependent mitochondrial apoptotic pathway involving caspase-3 activation. The results indicate that Roy-Bz targets drug resistant cancer stem cells, in HCT116 colon cancer cells, preventing tumor dissemination and recurrence. Moreover, these findings support a tumor suppressive function of PKCd in colon cancer. Overall, these results point to promising activators of PKCs with high potency and isoform-selectivity that may emerge from the exploitation of this new family of abietane diterpenoids [5]. Molecular docking studies are currently ongoing to further identify new selective abietane diterpenoids as new PKC modulators.

Speaker: Anna Meseguer Navarro
Novel biomarkers and therapeutic targets in clear cell renal cell carcinoma (ccRCC)

Renal cell carcinoma (RCC) is the third most prevalent urological cancer. It accounts for approximately 4% of all new cancer cases and the incidence rates for all stages have been rising steadily over the last 3 decades. Over 65.400 new renal cell carcinomas (RCCs) are annually detected in the USA, and 15,000 people will die from the disease. The clear cell RCC (ccRCC) is the most aggressive and common subtype and accounts for approximately 80% of all renal cancers.

Commonly asymptomatic , most RCCs are discovered incidentally on medical imaging. A great percentage of them may be treated by surgery, but one third of patients will present either with locally advanced tumor or with metastases, resulting in a 95% mortality rate. Moreover, one third of organ-confined cancers treated by nephrectomy develop metastasis during the follow-up. Conventional therapies have little effect on survival in patients with metastatic RCC. Until 2008, the 5-year survival rate for untreated metastatic RCC (mRCC) was 2%, and the median survival was approximately 8 months. In this scenario, our group was focused on the identification of novel diagnostic and prognostic biomarkers that could, eventually, become novel therapeutic targets for ccRCC patients. We first found that HAVRc-1, also known as KIM-1 or TIM-1, was up regulated in ccRCC tumors (60%) and had a role in cellular de-differentiation, scattering- and proliferating-related processes.
HAVRc-1 ectodomain was detected in the urine of ccRCC patients confirming its value as a noninvasive biomarker for early detection of ccRCC and the follow-up of metastatic patients, that was correlating with tumor grade and invasiveness. 

HAVRc-1 was expressed in ccRCC and papillary tumors and unexpectedly expressed in tubule cells of adjacent and distal unaffected counterparts of ccRCC suggesting that constitutive expression of HAVRc-1 in kidney could constitute a susceptibility trait for ccRCC tumor development. Clinical trials have confirmed this hypothesis since HAVRc-1 predicts RCC incidence up to 5 years prior to tumor diagnosis and constitutes a good marker for tumor relapse, after nephrectomy. We have also described that HAVRc-1 overexpression up-regulates IL-6 and activates the gp130/STAT-3/HIF-1 pathway in human ccRCC derived cell lines. This is an important pathway because: i) an IL-6 autocrine mechanism correlating with bad prognosis has been described in ccRCC; ii) STAT-3 regulates genes involved in tumor proliferation, apoptosis inhibition,immunosuppression and angiogenesis; iii) HIF-1A is a key element in promoting hypoxia-induced angiogenesis. pSTAT-3 activation studied in a tissue microarray (TMA) including 98 ccRCC patients with 5 to 10 years follow-up showed that pSer727-STAT3 is an independent prognostic factor of survival respect to classical clinic-pathologic features and constitutes a clinically relevant prognostic biomarker that correlates with ccRCC patient overall survival.
Development of targeted agents such as TKI inhibitors targeting angiogenesis and immunotherapies designed to block immune checkpoints such as cytotoxic T-lymphocyte– associated antigen 4 (CTLA4) and programmed cell death protein 1 (PD-1) has changed the choice of treatment for patients with mRCC. Although new therapies have improved the median survival period of patients with advanced ccRCC there are still many opportunities and challenges for non-responding patients or for those that develop resistance after treatment. We propose that HAVRc-1 and STAT-3 besides being good diagnostic and prognostic biomarkers might also represent novel targets for personalized treatment of ccRCC patients.

Speaker: Jazmine Arévalo (Oral Communication)
CM-728 as a potential and novel compound for metastatic clear cell renal cell carcinoma (ccRCC) treatment.

Clear cell renal cell carcinoma (ccRCC) is the most prevalent and lethal histological subtype of renal cell carcinoma (RCC). If detected early, partial or radical nephrectomy is the first-line treatment with an associated 5-year survival rate of 90%. In contrast, as ccRCC is generally asymptomatic, most patients are diagnosed in a more advanced and metastatic stage of the disease, for which the 5-year survival rate drops to 12%. At present, the first-line treatments for metastatic ccRCC are antiangiogenic agents that only stabilize the disease and are only effective in less than 50% of patients. One of the most prescribed is Sunitinib malate (Sutent®), a tyrosine kinase inhibitor (TKI) which acts partly by preventing the activation of the signal transducer and activator of transcription 3 (STAT3). Interestingly, an abnormal STAT3 activation has been correlated with poor prognosis and low overall survival in ccRCC patients. In consequence and based on the partial effectiveness of first-line treatments for advanced metastatic ccRCC, CEAMED SA is developing the compound CM-728 that has been shown to effectively inhibit STAT3 activation in several tumor cell lines. Therefore, the aim of this work was: i) to determine the sensitivity of human ccRCC derived cell lines (769-P, 786-O) to CM-728, ii) to analyze the effect of CM-728 on STAT3 activation (pTyr705) in these cell lines and, iii) to compare the effect of CM-728 with the first-line treatment Sunitinib, and with established JAK/STAT pathway inhibitors. Our results indicate that both human ccRCC derived cell lines tested (769-P and 786-O) are more sensitive to CM-728 than to Sunitinib and that CM-728 is a strong inhibitor of STAT3 activation in these ccRCC cell lines. In conclusion, our results suggest that CM-728 may have potential, as a novel compound for the treatment of metastatic ccRCC.

Speaker: Víctor Jiménez González (Oral Communication)
Preliminary screening for selective anticancer activity of Andalusian plants

According to the WHO data, lung cancer (along with bronchus and trachea cancers) is the sixth leading cause of global deaths in the last years. These cancers kill almost 2 million people every year. Despite the recent approval of new anticancer therapies, the tolerable doses of the current treatments are not yet enough to kill all cancer cells without not killing the normal health cells. As consequence, the treatments delay the progression of cancer but not the fatal ending. Therefore, it is necessary to find more selective anticancer drugs. Since plant kingdom is an important source of useful anticancer drugs, as paclitaxel used for treatment of lung cancer, we have started a random screening for selective anticancer activity of plants collected in Andalusia. Andalusia is autonomous community in southern Spain of high plant diversity and endemism. In this work, we show a preliminary results for selective anticancer activity of 32 extracts from 30 plants collected in western Andalucía. Using lung cancer cells A549 and non-malignant skin cells HaCaT, we demonstrated that some extracts were more cytotoxic and selective against cancer cells than the standard anticancer drug cisplatin. The highest anticancer selective activity was found on Daphne gnidium and Thymelaea hirsuta, two plants from Thymelaeaceae family. Further studies are needed to understand and evaluate the potential anticancer of these plants.

SESSION 12:
Oral Communications: Pain and Inflammation

Speaker: Eloi Franco Trepat
Amitriptyline, a new therapeutic tool to block innate immune responses in osteoarthritis patients

Background
Musculoskeletal pathologies, also known as rheumatic diseases, are rising their prevalence worldwide and depriving our society of the necessary welfare. Among musculoskeletal pathologies, osteoarthritis (OA) stands as the major rheumatic disease. OA is defined by joint-space narrowing due to progressive cartilage degradation, pain, and disability. In fact, the World Health Organisation (WHO) has included OA as one of the ten most disabling diseases in developed countries.
The activation of innate immune receptors, such as Toll-like receptor 4 (TLR4), by damage-associated molecular patterns (DAMPS), has been involved in chondrocyte-mediated inflammatory and catabolic responses. There are currently no available drugs aimed to block TLR4-mediated responses. Nonetheless, current studies in the use of amitriptyline, a drug already being employed in neuropathies such as depression, anxiety, and attention deficit, indicate that it might have anti-TLR4 activity.

Results
The effect of amitriptyline on inflammatory responses was observed upon stimulation of human OA chondrocytes with the TLR4 agonist LPS [100ng/ml]. After stimulation, several pro-inflammatory (LCN2, IL-6, and MCP-1) and pro-catabolic factors (MMP9, MMP13, and ADAMTS4) increased their mRNA expression. Consistent with these, the proteomic profile (MALDI/TOFF) from the stimulated cells showed enrichment in inflammation and catabolism. At the studied concentrations, amitriptyline did not affect chondrocytes viability and successfully reduced nitrite accumulation in the culture media.

Upon co-stimulation of these cells with amitriptyline [1µM] the observed responses were significantly inhibited: studied factors mRNA expression (↓95%) and studied pathway enrichment disappeared (Fig. 1).

Conclusions
We show that amitriptyline block TLR4 innate immune responses in human OA chondrocytes with no downside effects. The rheumatologists have a new therapeutic tool ready to manage innate immune responses in OA patients.

Speaker: Verónica Casadó-Anguera
Methadone is an analgesic with Low Addictive Side Effects due to its Weak Potency on Opioid-Galanin Receptor Heteromers

mu-Opioid receptor (MOR) agonists offer the most effective treatment for severe pain, making the search for a non-addictive opioid medication a high priority in medical sciences. MOR is a G protein-coupled receptor (GPCR) that is essential for opioid-induced analgesia, but also responsible for adverse effects, including respiratory depression, reduced gastrointestinal motility and euphoria that can lead to addiction. Thus, MORs mediate both the analgesic and addictive effects of opioids, responsible of the well-known opioid epidemic, which represents a severe public health crisis. The neuropeptide galanin acts as a modulator of neurotransmission in the CNS and in the peripheral nervous system, acting on three subtypes of GPCRs (Gal1R, Gal2R and Gal3R). Galanin is co-expressed with different neurotransmitters and co-released by the major ascending noradrenergic, serotoninergic, histaminergic and cholinergic systems. Biochemical and behavioral studies also indicated the functional presence of galanin and galanin receptors in dopaminergic areas, including the ventral tegmental area (VTA) and the nucleus accumbens (NAc), where they mediate an antagonistic effect on opioid reward. Here, we have found a significant pharmacodynamic difference between methadone versus morphine and fentanyl that entirely depends on the heteromerization of MOR with Gal1R, rendering a profound decrease in the potency of methadone. This explains methadone’s weaker ability to activate the dopaminergic system as compared to morphine and fentanyl and predicts a dissociation of therapeutic versus euphoric effects of methadone, that preferably binds to peripheral MOR which does not form heteromers with Gal1R. In conclusion, these results suggest that MOR-Gal1R heteromers mediate the dopaminergic effects of opioids and that opioids with selective low potency for the these heteromers, such as methadone, may have lower addictive side effects.

Speaker: Elia Álvarez Coiradas
Identification and Validation of two small Molecules Targeting the IL-17 Inflammatory Pathway

Interleukin 17 (IL-17) is a proinflammatory cytokine that not only plays a pivotal role in host defence against extracellular pathogens, but also acts as an immune checkpoint in several autoimmune diseases. As a result, the IL-17 family has become an attractive pharmacological target for inflammatory autoimmune diseases such as psoriasis, for which therapeutic antibodies have been approved for clinical use. 

Targeting IL-17 pathway with small molecules is feasible, although both novel methods and novel chemical matter are needed. We employed a virtual screening of our chemical library of 60 000 compounds to identify 67 potential ligands against IL-17 available structures. Then, we developed a biophysical label-free assay based on Dynamic Mass Redistribution technology, which allows the detection of the interaction between proteins and small molecules. We immobilized the extracellular domain of IL-17RA and we screened the ligands identified before in the label-free assay. 2 molecules sharing a common chemical scaffold were found active as potential binders with micromolar binding affinity and were confirmed by a Surface Plasmon Resonance assay. The functional activity of these ligands as inhibitors of the IL-17A-IL17RA interaction was first tested by a cytokine CXCL1 release assay based on blocking IL-17A proinflammatory stimulation of HT-29 colorectal cancer cells. Confirmation of this biological activity was tested in a keratinocyte cell line (HaCaT), showing that both compounds inhibit IL-17A proinflammatory activity as they decrease the release of CCL20 and IL-8. 

Combining biochemical and cell-based assays with structure-based design from this novel chemotype can facilitate the identification of compounds functionally targeting IL-17 inflammatory pathway. Further characterization of the efficacy of these molecules can facilitate their progress as preclinical therapeutic agents for psoriasis.

SESSION 13:
Oral Communications: Neuro/Psychopharmacology

Speaker: Valentín Ceña
Protective effect of Phosphorus dendrimers in a murine model of multiple sclerosis

PROTECTIVE EFFECT OF NEUTRAL PHOSPHORUS DENDRIMERS IN A MURINE MODEL OF MULTIPLE SCLEROSIS
I. Posadas 1,2, L. Romero-Castillo 1,2, V. Ceña. 1,2
1. Unidad Asociada Neurodeath, Universidad de Castilla-La Mancha, Albacete, Spain
2. CIBERNED, ISCIII, Madrid

Background
Inflammation is a physio-pathological process raised in response to different exogenous and endogenous stimulus. Following the recognition of these stimuli, inflammatory cells including macrophages and lymphocytes, activate several pathways allowing the inflammatory response to eliminate the disturbing element, and resolve the inflammation. However, in certain pathologies, inflammation is not resolved resulting in chronic inflammatory diseases such as arthritis rheumatoid or multiple sclerosis. Phosphorus dendrimers have recently emerged as interesting immunomodulatory nanoparticles able to modulate macrophages polarization and reduce the peripheral inflammatory response.
Material and Methods.
Cortical neurons or astrocytes were obtained from C57BL/6j mouse embryonic fetuses 17 days or from 1-5 days old pups, respectively. Lymphocytes were obtained from spleen and thymus from adult C57BL/6j mice. Lack of toxicity was studied by MTT, LDH and proliferation assays.
Lymphocytes-derived cytokines were determined by ProcartaPlex Multiplex immunoassay. Experimental Autoimmune Encephalitis (EAE) was induced by standard procedures sensitizing the mice with a MOG peptide. The animal’s clinical score was determined using a generally accepted scale ranging from 0 to 5.


Results
Phosphodendrimers (generations 3 and 4) displayed a good security profile in all cells studied. Both prevented CD3: CD28-induced cytokine production in a concentration dependent-manner without affecting lymphocytes viability. G3b and G4b dendrimers significantly prevented MOG-induced disability in the experimental model of EAE, displaying a potency similar to the reference compound fingolimod.
Conclusion.
Neutral phosphorous dendrimers display promising features for the treatment of autoimmune diseases.

Speaker: Alejandra Palomino Antolín
NLRP3 inflammasome inhibition protects the neurovascular unit, reduces infarct volume and inflammation in cerebral ischemia.

Cerebral ischemia is the third cause of death and the main cause of adult disability worldwide. Currently the only pharmacological treatment for acute ischemic stroke is intravenous tissue plasminogen activator (tPA). However, only 10% of patients benefit from tPA administration, due to its limited therapeutic window and the risk of intracerebral hemorrhage. Inflammation in ischemic injury is crucially mediated by NLRP3, a key component of inmune system. In this study, we investigated the role of NLRP3 in post-ischemic inflammation, using MCC950, a potent inhibitor of NLRP3 inflammasome. For that purpose, we used transient middle cerebral artery occlusion (tMCAO) during 1 hour in mice as a model of cerebral ischemia. Administration of MCC950 1h after reperfusion reduced infarct volume in a dose-dependent manner (1, 3, 10 mg/kg). As a clinical outcome parameter, MCC950 at 3 mg/kg improved neuro-motor function and reduced expression of different pro-inflammatory cytokines (IL-1β, TNF-α, IL-6), chemokines (CCL2) and NLRP3 inflammasome components (pro-caspase-1, NLRP3, pro-IL-1β). We observed that tMCAO produced Blood Brain Barrier (BBB) disruption that was improved in animals treated with MCC950 (3 mg/kg). In MCC950-treated animals, we observed a functional recovery of endothelial proteins that forms the tight junctions of BBB (VE-cadherina, Claudin-5, ZO-1), together with a reduction of the expression of adhesion molecules (ICAM, VCAM) and matrix metalloproteases (MMP9), that leads to a lower peripheral inmune cells infiltration in the infarction area (Ly6G+ neutrophils, leukocytes CD45high, microglia CD11+, Ly6C+ macrophages). From these results we can conclude that inhibition of NLRP3 inflammasome with MCC950 significantly reduces infarct volume and improves neuro-motor function and protects the neurovascular unit by improving the BBB integrity through stabilization of the tight junctions. Hence, the inhibition of NLRP3 may be a promising target in cerebral ischemia.

Speaker: Maria Julia García-Fuster
Cannabidiol Antidepressant-Like Effects in Rats: Decreased Sensitivity in Adolescent versus Adult Rats

Cannabidiol is a non-psychoactive phytocannabinoid with great therapeutic potential in diverse psychiatric disorders, however its beneficial effects have been mainly ascertained in adult rats. This study aimed at comparing the antidepressant-like effects induced by cannabidiol in adolescent and adult rats and the possible parallel modulation of hippocampal neurogenesis. Male Sprague-Dawley rats were repeatedly treated with cannabidiol (3, 10 and 30 mg/kg) or vehicle (1 ml/kg) during adolescence (postnatal days, PND 27-33) or adulthood (PND 141-147) and exposed to 3 consecutive tests, 2-3 days apart (forced-swim, open field, sucrose preference) that quantified different aspects of affective-like behavior (behavioral despair, anxiety- and hedonic-like responses). Cannabidiol induced differential effects depending on the age and dose administered when compared to control rats: (1) cannabidiol (30 mg/kg) decreased body weight only in adult rats following 6 (-34±15 g, p<0.05) and 7 (-40±15 g, p<0.05) days of treatment; (2) cannabidiol improved behavioral despair in adolescent and adult rats, but with a different dose sensitivity (10 vs. 30mg/kg), and with a different extent (2 vs. 21 days post-treatment: -59±19 sec immobile, p<0.05 and -55±20 sec immobile, p<0.01 respectively); (3) cannabidiol did not modulate anxiety-like behavior at any dose tested in adolescent or adult rats; and (4) cannabidiol induced prohedonic-like effects in adult rats following 10 mg/kg (+50±18 g, p<0.05) and 30 mg/kg (+32±5 g, p<0.05). Our findings support the notion that cannabidiol exerts antidepressant- (improved behavioral despair and prohedonic) and anorexigenic-like effects in adult rats, and demonstrate a decreased sensitivity when administered in adolescent rats. Moreover, since cannabidiol did not modulate hippocampal neurogenesis (cell proliferation and early cell survival) in adolescent or adult rats, the results revealed potential antidepressant-like effects induced by cannabidiol without the need of regulating hippocampal neurogenesis. Supported by ‘Delegación del Gobierno para el Plan Nacional sobre Drogas’ (2016/002, MSSSI) and by RTA-RD16/0017/0010.
Key words: antidepressant; age; adolescence; rat

Speaker: Marta Cimadevila
Essential Role of C148-C227 Disulfide Bridge in Human 5-HT2A Receptor Functionality and Trafficking

G Protein Coupled Receptors extracellular domains are emerging as a determining factor in receptor functionality, not only for orthosteric ligands, but also as an allosteric modulation site (Wooley MJ, et al., 2017). The recent crystallization of serotonin 2A (5-HT2A) receptor showed a critical role of extracellular loop 2 (ECL-2) in receptor selectivity. This finding is in agreement with previous studies, where dithiothreitol was used to nonspecifically break a conserved disulfide bridge between C148 at transmembrane domain 3 (TM-3) and C227 at ECL-2 (Iglesias A, et al. 2017).
Our hypothesis is that the C148-C227 disulfide bridge in 5-HT2A receptor may be critical for ligand binding, receptor activation and trafficking. Thus, we aimed to generate three constructions incapable of maintaining this bridge and study their binding, function and subcellular expression.
The two cysteines involved in the aforementioned disulfide bridge were mutated into alanines, obtaining three constructs: pcDNA5/FRT/TO myc-5-HT2AC148A-eYFP (C148A), pcDNA5/FRT/TO myc-5-HT2AC227A-eYFP (C227A) and pcDNA5/FRT/TO myc-5-HT2AC148A/C227A-eYFP (C148A/C227A). These three constructions, together with the parental one (pcDNA5/FRT/TO myc-5-HT2AeYFP, WT) were employed to generate four stable cell lines that were characterized by means of binding and functional assays. For subcellular localization studies, cell nucleus and the endoplasmic reticulum were stained with fluorescent probes, and images were obtained in Operetta High Content Imaging System (Perkin Elmer).
When compared with WT cell line, C148A, C227A and C148A/C227A mutants were unable to bind [3H]LSD and to respond to (±)DOI neither when measuring IPs accumulation, nor calcium mobilization. Furthermore, all the mutants showed around a 50% decrease of expression of receptor in the cell membrane.
In conclusion, these results show that the disulfide bridge formed between TM-3 and ECL-2 is critical to maintain human 5-HT2A receptor proper conformation for ligand binding and receptor activation, but also to facilitate receptor trafficking to cell membrane.

Closing Lecture:
Resolution Pharmacology

Speaker: Mauro Perretti
Resolution Pharmacology

Inflammation is a defensive response we mount against pathogens and injurious agents.  While a properly regulated inflammatory response is integral to physiology and well-being, a loss of regulation can lead to fibrosis and other chronic diseases. Over the last two decades, we have proposed that Important cues to correct persistent inflammation which typifies virtually all chronic pathologies may derive by studying how physiological inflammation resolves. As such we and others have identified specific pro-resolving mediators and receptors that are engaged for an efficient resolution. In this context, specific G-protein coupled receptors signal on macrophages and neutrophils to evoke pro-resolving and tissue-protective effects. More recently, the importance to signal resolution in stromal cells is emerging as necessary for an efficient resolution. Altogether, we propose that this wealth of scientific knowledge makes time ready to harness the biology of resolution for therapeutic innovation. The development of novel strategies to inform new treatments for chronic inflammatory diseases will establish a new branch of pharmacology we term ‘Resolution Pharmacology’. The flexibility of the biology of resolution can provide opportunities for monotherapies or poly-pharma approaches for better clinical management of complex diseases.