# Role of inflammatory processes in depression

Lucile Capuron

► To cite this version:

Lucile Capuron. Role of inflammatory processes in depression. 37. SEF National Meeting with guest society: the British Pharmacology Society, Jun 2017, Barcelone, Spain. Basic and Clinical Pharmacology and Toxicology, Volume 121, Supplement 2, Special Issue SI, 2017, Basic and Clinical Pharmacology and Toxicology. 10.1111/bcpt.12824. hal-01608055

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Submitted on 2 Jun 2020

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# PLENARY SPEAKERS

## PL-1
### ROLE OF THE ENDOCANNABINOID SYSTEM IN FOOD-INDUCED ADDICTIVE-LIKE BEHAVIOR

Maldonado R.

Laboratorio de Neurofarmacología, Universitat Pompeu Fabra, Parc de Recerca Biomèdica de Barcelona, Barcelona, Spain

An increasing perspective conceptualizes obesity and overeating as disorders related to addictive-like processes that could share common neurobiological mechanisms. We aimed at validating an animal model of eating addictive-like behavior in mice, based on the DSM-5 substance use disorder criteria, using operant conditioning maintained by highly palatable chocolate-flavored pellets. For this purpose, we evaluated persistence of food-seeking during a period of non-availability of food, motivation for food, and perseverance of responding when the reward was associated with a punishment. This model has allowed identifying extreme subpopulations of mice related to addictive-like behavior. We investigated in these subpopulations the Epigenetic and proteomic studies have allowed to identify a significant decrease in DNA methylation of CNR1 gene promoter in the prefrontal cortex of addict-like mice, which was associated with an upregulation of CB1 protein expression in the same brain area. The pharmacological blockade of CB1 receptor during the late training period reduced the percentage of mice that accomplished addiction criteria, which is in agreement with the reduced performance of CB1 knockout mice in this operant training. Proteomic studies have identified proteins differentially expressed in mice vulnerable or not to addictive-like behavior in the hippocampus, striatum, and prefrontal cortex. The use of DREADD techniques in this model has now allowed identifying the crucial role of the prefrontal cortex in the development of eating addictive-like behavior. This model provides an excellent tool to investigate the neurobiological mechanisms underlying eating addictive-like behavior.

## PL-2
### GPCR EXPRESSION IN HEALTH AND DISEASE IDENTIFIES NEW THERAPEUTIC TARGETS

Insel P.A., Sriram K., Wiley S., Michkov A., Gorr M., Chinn A., Chang S., Liang W., Corriden R.

Departments of Pharmacology and Medicine, University of California San Diego, La Jolla, CA, USA

G protein-coupled receptors (GPCRs) are the most widely targeted class of FDA- and EMA-approved therapeutics but only a small portion (~15%) of known GPCRs are currently targeted with such drugs. We have tested the hypothesis that individual cell types endogenously express previously unrecognized GPCRs that may be drug targets. Our recent efforts have focused on human cells and on defining differential expression of GPCRs on cells from patients with certain diseases, including pulmonary arterial smooth muscle cells/pulmonary arterial hypertension, lung and cardiac fibroblasts/lung and cardiac fibrosis and pancreatic cancer compared to control cells. To test our hypothesis, we have used unbiased approaches (Taqman GPCR arrays and RNA-seq), mining of publicly available datasets (such as The Cancer Genome Atlas, TCGA), and validation studies to assess GPCR signaling and functional activity. We find that most cell types express >100 different GPCRs. Little or no prior functional data exist in such cells for many of the highest expressed GPCRs, numerous of which are “orphans” (without known physiologic agonists). Differentially expressed GPCRs in the disease settings can alter cell function. A major focus has been on pancreatic ductal adenocarcinoma (PDAC) tumors and cells and

pancreatic cancer associated fibroblasts (PCAFs). We have identified two orphan GPCRs (Orphan 1 and Orphan A) with high expression in PCAFs and PDAC cells, respectively, and at least 2-fold higher expression in >90% of PDAC tumors (in TCGA) compared to normal pancreas. Orphan 1 and Orphan A regulate functional activity that influences the malignant phenotype. Overall, our results imply that previously unrecognized GPCRs contribute to several diseases and thus may be novel, druggable targets. Moreover, our approach should be readily applicable to other cell types and disease settings. (Supported by NIH grants R21CA202608, R21AG053568, R56AI110505, T32GM007752, T32HL007444, T32HL134632, DoD grant W81XWH-14-1-0372 and a contract with Bristol Myers Squibb).

## PL-3
### CANCER EPIGENETICS: GENES AND DRUGS

Esteller M.

Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), 08908 L'Hospitalet de Llobregat, Barcelona, Catalonia, Spain

For the last 25 years an increasing amount of evidence has shown the relevance of epigenetics in cell biology and tissue physiology, being DNA methylation aberrations in cancer the flag-ship for the recognition of its disturbance in human diseases. From the candidate gene approaches, new powerful technologies such as comprehensive DNA methylation microarrays and whole genome bisulfite sequencing has recently emerged that have reinforced the notion of epigenetic disruption in the crossroad of many sickness. From the poster-boy cases of MGMT and GSTP1 hypermethylation in the prediction of alkylating drug response and prostate cancer detection, respectively, to the personalized treatment of leukemia with small molecules targeted to fusion proteins involving histone modifiers such as DOT1L and MLL, the field has walked a long path. The current talk will focus in the epigenetic profiling, basically at the level of DNA methylation and histone modifications, that is starting to provide clinical value in the diagnosis, prognosis and prediction of response to drug therapies, with an emphasis in neoplasia, but without forgetting the novel advances in other human disorders. For cancer, we have already a wide view of the undergoing DNA methylation events that expand beyond classical promoter CpG islands of tumor suppressor genes and we have a growing list of mutated chromatin remodeler genes that contributes to the tumorigenesis process. It is time to apply this knowledge in practical clinical situations like the diagnosis of cancers of unknown primary, the screening of malignancies in high-risk populations or a biomarker selection of the patients that should receive treatment with epigenetic drugs.

## PL-4
### A NOVEL PHARMACOLOGICAL PARADIGM FOR CHRONIC PAIN

Montiel A.F.

Instituto de Biología Molecular y Celular. Universidad Miguel Hernández, Elche, Spain

Pain is the most common complaint for which patients seek treatment from a physician and it has been estimated that nearly half a billion cases are diagnosed each year. Both acute and chronic pain have a major impact on many quality-of-life measures¹. Chronic pain is
suffered by up to 20% of the adult European population and its inci-
dence is rising as the population ages. For over 50% of these individu-
als treatment remains unsatisfactory. Despite the prevalence of
undertreated chronic pain, the pharmacological armamentarium to pre-
vent or reduce it is surprisingly limited, mainly due to our poor under-
standing of the genetic, molecular and cellular mechanisms underlying
various pain syndromes, along with the inter-individual variation. A
large focus has been directed to modulate peripheral receptor operated
signalling in nociceptors, developing high affinity modulators. How-
ever, these approaches have not rendered yet the expected clinical ben-
efits. We have alternatively focused our pharmacological approach on
modulating the disease-induced expression of neuronal receptors. A
genomic study revealed that thermoreceptors such as TRPV1 are
recruited to the neuronal surface by inflammatory and algesic agents,
leading to nociceptor sensitization. Abrogation of TRPV1 recruitment
notably reduces neuronal sensitization and produces strong anti-noci-
ception in models of inflammatory and neuropathic pain. The most
advanced compound targeting this mechanism is currently in clinical
development for chronic pain. Funded by MINECO and GVA (PRO-
METEO Program).

PL-5
PHARMACOLOGY LEAPING OVER THE GAP: BRINGING
TRANSFORMATIONAL MECHANISMS TO DRUG
DISCOVERY

Brea J.M., Dominguez E., Loza M.I.

Grupo BioFarma, Facultad de Farmacia, Instituto de Farmacia
Industrial, Centro de investigación CIMUS, Universidad de Santiago
de Compostela, Santiago de Compostela, Spain

Open Innovation has transformed early drug discovery in a highly
multidisciplinary and delocalized scenario, including all the partners
sharing knowledge in real time through the entire health ecosystem.
The changing face of drug discovery could facilitate to apply, in a
holistic way, the pharmacological research path to transformational
biomedical research programs. Pharmaceutical companies have created
new departments of open innovation in which worldwide best experts
are connecting the research on the mechanisms of disease with their
pipelines. It is a new world to create ‘real’ innovation in drug discov-
ery.

This represents a great opportunity for experimental pharmacology to
joint to the new scenario, connecting biomedical public research in the
mechanisms of disease with clinical, biotechnological and pharmaceuti-
cal companies' experts and patients. This change of scientific/technical
perspective also implies the creation of innovative figures and models
of scientific, legal and financial management.

It is a difficult challenge for this new era and many ingredients like
know-how, vision, serendipity, scientific management, in addition to
the excellent science are required. For instance, in Spain from more
than 1000 programs analyzed in Venture Capital, only 20 were
advanced. In any case, deals are coming in the last years (for instance
from Esteve, Almirall, Orizon, PaloBiofarma...).

Different public-private initiatives aimed at developing transforma-
tional pharmacological programs that yield drug will be discussed.
Essentially, there are two perspectives: i) to look for the best science
across the world in the area of interest of the pharmaceutical compa-
nies and incorporate it through the creation of joint laboratories or its
absorption in the case of small startups; ii) to search for existing inno-
vative models, already oriented towards therapeutics and to potentiate

their innovative trough collaboration/funding, with independent agree-
ments without absorption.

The experience of our lab in both models will be illustrated trough: i) Esteve-USC Joint Research Unit, an effort co-funded by the Galician Government that has launched a special program. Esteve has consolided its collaboration with the USC by implementing a joint research unit that works as an incubator of in vitro applied pharmacology within compact team sharing public/private research, with a successful outcome both in new methodologies and new chemical entities characterized through that methodologies; ii) Innopharma Platform/Kærtor Foundation, it arises from our experience from 2 years of intensive work with a committee of international experts in open innovation, drug discovery and financial models. Its mission is to manage the reciprocal transfer of private/public knowledge in a 'totipotent model' devoted to leaping the gaps in connection/translation in the whole process of drug discovery. Outcomes include new pharmacology applied programs, connected to 25 agreements with principal investigators/ technology transfer offices, 5 agreements with pharmaceutical companies and 1 program with clinical proof of principle finished.

In conclusion, open innovation in drug discovery connecting public
and private research represents a challenge but also a great opportunity
for experimental pharmacology to move the most innovative disease
mechanisms to drugs for unmet medical needs.

PL-6
PRESENT AND FUTURE TREATMENT OF ALZHEIMER'S
DISEASE

Nordberg A.

Department of Neurobiology, Care Sciences and Society, Translational Alzheimer Neurobiology, Karolinska Institutet, Stockholm, Sweden

Alzheimer's disease is the most common form of dementia disorder and poses a great challenge for the society. Owing to a steep future increase in the number of elderly worldwide there is a strong urge to find effective targets for treatment of diseases as AD. Drugs with symptomatic effects such as the cholinesterase inhibitors donepezil, rivastigmine and galantamine and the NMDA antagonist memantine are today clinical standard therapy in AD. How should we find disease modifying drugs or even preventive/protective therapy? By modern molecular imaging techniques it is now possible to visualize and measure in vivo proteinopathies already described by Dr Alois Alzheimer as amyloid plaques and neurofibrillary tangles in AD brains. The development of new AD therapies has for more than a decade been dominated by anti-beta amyloid therapies. These include monoclonal antibodies in passive immunizations as well as vaccine in active immunizations. Thus several anti-beta amyloid humanized monoclonal antibodies including bapineuzumab, solanezumab, crenezumab and others have been tested in large clinical trials at different stages of AD but have not met the pre-determined endpoints to alter the course of AD. The beta-amyloid antibody Aducanumab has shown in a phase 2 study of early AD a dose related decrease in amyloid plaque load in brain measured by PET as well as some effects in cognitive tests. Other anti-beta amyloid approaches as the gamma secretase inhibitors failed and the beta amyloid cleavage enzyme 1 (BACE1) inhibitors have still to demonstrate clinical efficacy. Other new interesting approaches are anti tau vaccines. Other strategies are focusing on nerve growth factors (NGF), novel nicotinic receptor agonists and drugs interfering with inflammatory processes in brain.

**Key words:** Alzheimer's disease, symptomatic drugs, disease modifying therapies, immunizations therapy, beta-amyloid, tau, acetylcholine
# SYMPOSIA 1: PHARMACOLOGY AND TOXICOLOGY OF DRUG TRANSPORTERS: RECENT ADVANCES

## S1-1
PHARMACOGENOMICS AND EPIGENETIC ALTERATION OF MEMBRANE TRANSPORTERS

Schwab M.¹,², Fisel P.¹, Schaeffeler E.¹

¹Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart; ²Department of Clinical Pharmacology, University Hospital Tuebingen, Tuebingen, Germany

Interindividual variability of expression and function of membrane transporters with consequences on drug response is not only affected by genetic factors but could be also explained by epigenomics (DNA methylation, histone modifications, regulation by miRNA). DNA hypermethylation results in gene silencing by direct inhibition of transcription-factor binding or by recruitment of methylated DNA-binding proteins. There is proof of principle for the clinical value of methylation markers for classification, prognosis and prediction of therapeutic response, and tissue-specific methylation alters the expression of selected ADME genes (Fisel et al. Clin Pharmacol Ther 2016). Systematic knowledge, however, related to human uptake transporters is still limited. Therefore, we investigated the impact of DNA methylation for selected SLC transporters on whether tissue specific expression in normal vs. corresponding tumor tissues is regulated by core gene regions with clinical consequences. DNA methylation regulates gene expression of an important SLC transporter which is the monocarboxylate transporter 4 (MCT4), a metabolic target in tumor biology because it mediates lactate transport across membranes resulting in antiapoptotic effects. MCT4 protein is significantly overexpressed in >85% of renal cancers (ccRCC), and DNA methylation at specific CpGs in the SLC16A3 promoter correlates significantly inverse with MCT4 expression in ccRCC resulting in adverse cancer-specific outcome. Apart from the prognostic potential for patient outcome, the transcriptional regulation of MCT4 by DNA methylation offers novel and attractive opportunities for therapeutic intervention by specific silencing of MCT4 and subsequent targeting of lactate efflux in ccRCC. A second example is the expression of the drug uptake transporters OCT1 in human liver and OCT2 in human kidney which is determined by DNA-methylation. It has been shown that DNA methylation of the hepatic SLC22A1 is associated with downregulation of SLC22A1 in hepatocellular carcinoma (HCC) which might be a novel biomarker for HCC diagnosis and prognosis. Since platinum drugs are substrates of SLC22A1, the modulation of gene expression by pretreatment with demethylating agents may offer novel therapeutic options for anticancer therapy. The large-scale, systematic epigenomic equivalents of GWAS, termed epigenome-wide associations studies (EWAS), are promising tools to determine specific drug-related phenotypes attributable to interindividual epigenomic variation. Thus, there appears to be a promising potential of epigenomics of transporter proteins in determining interindividual variation in drug response and in serving as targets for epidrug intervention.

The work is supported in part by the Robert Bosch Stiftung, Stuttgart, Germany.

## S1-2
PBPK MODELLING OF DRUG TRANSPORTERS TO FACILITATE INDIVIDUALIZED DOSE PREDICTION

Galetin A.

Centre for Applied Pharmacokinetic Research, University of Manchester, UK

Physiologically-based pharmacokinetic (PBPK) modelling is increasingly used as a decision-making tool in different stages of drug development. This approach is particularly important for prediction of complex drug-drug interactions involving interplay between multiple transporters and/or enzymes in different tissues. Current challenges in the translation of transporter kinetic data to in vivo within PBPK paradigm will be illustrated by simvastatin model that incorporates complex lactone-acid inter-conversion. Changes in both simvastatin forms under different clinical scenarios (drug co-administration, genetic polymorphism in OATP1B1 transporter) were simulated at the systemic level, but also in the tissues of interest selected either due to efficacy/interaction concerns (e.g., liver) or safety (e.g., muscle). Presentation will also illustrate areas where PBPK modelling is expected to inform drug labelling in the future (e.g., special populations), highlighting current knowledge gaps from the transporter perspective.

## S1-3
ABC TRANSPORTERS IN DRUG-INDUCED LIVER INJURY

Manautou J.E.

Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut, USA

Drug-induced liver injury (DILI) continues to be prominent problem in drug development and patient care. Although our knowledge of DILI mechanisms is incomplete, multiple cellular events and their molecular mediators contributing to DILI have been described. Among these, hepatobiliary transporters, which are key genetic determinants of drug pharmacokinetics and pharmacodynamics, have emerged as important contributors of DILI outcome. DILI is also known to alter the expression and function of drug transporters. The physiological consequences of such an adaptive response are not well understood. We have demonstrated that DILI resulting from acetaminophen (APAP) treatment leads to tolerance to subsequent hepatotoxicant exposure. Our work indicates that this adaptive process is a general response to tissue injury and not a drug-specific event. We have further investigated whether changes in the expression and function of drug transporters contribute to the development of tolerance to APAP hepatotoxicity, with emphasis on the multidrug resistance protein 4 (Mrp4, ABCC4). Research work to be discussed include studies investigating the role of ABCC4 in compensatory cell proliferation following DILI and other models of liver tissue loss and regeneration. This presentation will also highlight the utility of knockout animal models in defining the role of drug transporters in DILI and related liver diseases.

**Key words:** acetaminophen; drug transporters; liver; drug resistance
S1-4
SCIENTIFIC PERSPECTIVE ON THE EVALUATION OF
TRANSPORTER-BASED DRUG-DRUG INTERACTIONS

Huang S.-M.

University of Maryland, College Park, MD, USA

Alterations in transporter activities can contribute to variability in phar-
macokinetics, pharmacodynamics and efficacy and safety of drugs.
Drug transporter-based drug-drug interactions have been increasingly
evaluated during drug development (1-5). Regulatory agencies have
issued guidance documents (6-8) to suggest when, what and how DDI
be evaluated during drug development and labeling implications of
these studies. This presentation will discuss scientific issues in the use
of in vitro, in vivo and in silico methods of assessing these interactions
and will include the following.

1. Various in vitro criteria that have been used to determine whether in vivo studies for P-gp, OATP1B1, OAT1/3, and OCT2-based interactions are needed

2. Availability of substrates and modulators to use in transporter-based DDI in vitro and in vivo (9)

3. The need to consider tissue concentrations in interpreting systemic exposure data from DDI studies.

4. How physiologically-based pharmacokinetic modeling (PBPK) and simulations have been used in the evaluation of DDI- applications and limitations (10-12)

5. Examples of complex DDI (13–14)

6. The need for continued collaborative work to address transporter-
based DDI

**References:**

1. Giacomini, Huang, Tweedie, Benet, Brouwer, Chu, Dahlin, Evers, Fischer, Hillgren, Hoffmaster, Ishikawa, Keppler, Kim, Lee, Niemi,

Polli, Sugiyama, Swan, Ware, Wright, Yee, Zamek-Gliszczynski,
Zhang, Nat Rev Drug Discov March 2010

2. Huang and Woodcock, Nat Rev Drug Discov. March 2010

3. Giacomini and Huang, Editorial, Clin Pharmacol Ther. July 2013

4. International Transporter Consortium workshop 3, American Society for Clinical Pharmacology and Therapeutics annual meeting, March 13–14, 2017, Washington DC

5. Tweedie D, et al., Clin Pharm Ther, July 2013

6. FDA draft DDI guidance 2012 http://www.fda.gov/Drugs/Guidance
ComplianceRegulatoryInformation/Guidances/ucm064982.htm

7. EMA DDI guidance 2013

8. PMDA guidance 2014

9. FDA drug development and drug interactions website: http://www.fda.gov/drugs/developmentapprovalprocess/developmen tresources/druginteractionslabeling/ucm080499.htm

10. Huang, Abernethy, Wang, Zhao, Zineh, J Pharm Sci 102(9):2912-2923 (2013)

11. Wagner, C., P. Zhao, Y. Pan, V. Hsu, J. Grillo, S. M. Huang and V. Sinha (2015). "Application of Physiologically Based Pharmacokinetic (PBPK) Modeling to Support Dose Selection: Report of an FDA Public Workshop on PBPK." CPT Pharmacometrics Syst Pharmacol 4(4): 226-230.

12. FDA Advisory Committee March 15, 2017- FDA Pharmaceutical Science and Clinical Pharmacology Advisory Committee meeting, March 15, 2017, Washington DC. https://www.fda.gov/Advi soryCommittees/Calendar/ucm535513.htm Last accessed February 20, 2017

13. OLYSIO (simeprevir) Clinical Pharmacology Review March 2013, Pages 88, 91 http://www.accessdata.fda.gov/drugsatfda_docs/nda/2013/205123Orig1s000TOC.cfm

14. Snoeys J, et al Clin Pharmacol Ther 2016.
# SYMPOSIA 2: PAIN

## S2-1
PHARMACOLOGICAL ASSOCIATIONS IN PAIN. A REAL-LIFE SCENARIO

*Mayoral V.*

Pain Management Unit, Anaesthesiology Department, University Hospital of Bellvitge-IDIBELL, L'Hospitalet de Llobregat, Barcelona, Spain

Very few studies and less clinical trials with analgesic drugs reproduce what is the real clinical practice where patients are taking multiple drugs (analgesic and not), either under prescription but also on OTC. Synergistic analgesia but also serious adverse reactions have been described with the associations of different pure analgesics drugs but also with co-analgesics. Except pure opioids agonists, all other analgesics have a ceiling effect, but high and increasing doses are also associated with a higher probability adverse reactions, mainly neurological and gastrointestinal ones. The successful concept of multimodal analgesia, already a standard of care in postoperative pain, takes advantage of the opioid sparing effect of other analgesics like NSAIDs, Paracetamol, Metamizole, local anaesthetics, ketamine and even Gabapentinoids. This concept has been extended to the clinical management of other kinds of acute pain as well as chronic pain, either neuropathic or nociceptive ones. Although the rationale of this approach seems logical even in chronic pain, safety concerns plays a major role in a context of less supervision as home care. The main common adverse reactions due to combinations that deserve supervision are: serotonin syndrome with the association of opioids such as Tramadol and antidepressants (TCA or SNRI) also indicated in neuropathic pain; the combined use of Antidepressants and NSAIDs are generally believed to each increase the risk of abnormal bleeding, even intracranial haemorrhage; the liver toxicity of Paracetamol and Duloxetine; and the potential of serious arrhythmias due to a long QTc that may be elongated with the combination of several antiepileptic drugs or combinations of Methadone.

## S2-2
IMPLICATION OF NORADRENALINE IN PAIN AND ITS EMOTIONAL CONSEQUENCES

*Berrocoso E.*

Psychobiology Area, University of Cádiz, Cádiz, Spain

The activation of the descending noradrenergic pathway has been implicated in the analgesic effects of antidepressants and anticonvulsants. However, many patients fail to achieve adequate analgesia with these medications or they do not tolerate them. Thus, it is possible that pain produces specific neuroplastic changes that modify the influence of noradrenaline. Furthermore, little is known about the role of the ascending noradrenergic projections on the activity of corticolimbic structures where the emotional aspects of pain are processed.

During this Workshop, novel data will be presented that has been obtained through the combined use of chemogenetics and electrophysiological tools, providing new insights into the noradrenergic system and the amygdala circuits mediating the effects of chronic pain, and pain-induced anxiety. I will show that direct activation of the noradrenergic locus coeruleus (LC) can produce both pro- and anti-nociceptive effects. In addition, the noradrenergic circuit that mediates stress-induced anxiety and aversion will be described, and I will show that the noradrenergic system is a critical hub for the development of the anxiodepressive consequences of long-term pain. These data stimulate

interest in novel therapeutic options that modulate the noradrenergic and amygdala circuits.

**Key words:** noradrenaline; locus coeruleus; amygdala; pain

## S2-3
SIGMA-1 RECEPTOR BLOCKADE UNMASKED PERIPHERAL OPIOID ANALGESIA

*Baeyens J.M.*

Department of Pharmacology and Neuroscience Institute, Faculty of Medicine, University of Granada, Granada, Spain

Opioid receptor agonists constitute one of the pain treatment milestones, but frequently produce centrally-mediated side effects, such as respiratory depression or drug addiction. Ways to minimize these side effects can be to use blood brain barrier-impermeable opioid agonists or to inhibit peripheral brakes for opioid analgesia. Sigma-1 receptor (σ1R) is a chaperone able to interact with several channels and receptors, including mu-opioid receptors. σ1R blockade, either pharmacologic or genetic (σ1R knockout, S1KO), markedly enhance antinociception produced by systemic (sc) administration of opioid agonists. Since σ1R is highly expressed on primary afferent neurons, we hypothesized that at least a part of such enhancement might be produced peripherally. We found that intraplantar (ipl) administration of several opioid receptor agonists (morphine, oxycodone, buprenorphine) was devoid of effect against a mechanical nociceptive stimulus (paw pressure) in control wild-type (WT) mice, but produced a dose-dependent antinociception in S1KO mice. Moreover, σ1R antagonists (S1RA, BD-1063), injected either locally (ipl) or systemically (sc), dose-dependently enhanced the antinociceptive effect of ipl injected opioids. The enhancement was locally-produced, because it did not appear when the opioid agonist was injected in a paw and the σ1R antagonist in the contralateral paw, and was due to σ1R blockade, because it was reversed by a σ1R agonist (PRE-084). These mechanisms are relevant in pathological conditions such as inflammatory pain. During inflammation induced by ipl carrageenan immune cells infiltrate the lesioned tissue, where they release endogenous opioids (such as β-endorphin). In this model, σ1R antagonists inhibited mechanical hyperalgesia and their antihyperalgesic effect was reversed by naloxone-methiodide (a peripherally restricted opioid receptor antagonist) and by local administration of monoclonal antibody 3-E7, which recognizes an N-terminus sequence (Tyr-Gly-Gly-Phe) of most endogenous opioid peptides. Therefore, σ1R is a brake for endogenous and exogenous opioid-induced antinociception and represent an adequate drug-target to enhance peripheral opioid antinociception.

**Key words:** Sigma-1 receptor agonists, opioid agonists, endogenous opioids, inflammatory pain

## S2-4
ANIMAL MODELS OF PAIN: BEYOND THE EVALUATION OF REFLEXES

*Portillo-Salido E*¹, *de la Puente B*¹, *Cobos E.J.*², *Zamanillo D*¹, *Merlos M*¹

¹Drug Discovery and Preclinical Development, Esteve, Barcelona, Spain; ²Department of Pharmacology, School of Medicine, University of Granada, Granada, Spain

The discovery of novel targets and the development of better analgesics critically depend on the validity of the preclinical animal
models. Preclinical testing of pharmacological agents with a potential
for pain modulation has almost exclusively consisted of reflex tests.
However, clinical testing of candidate analgesics asses not only
changes in pain intensity but also in physical and emotional function-
ing. In fact, the analgesic-induced recovery of normal physical and
emotional functioning is considered at least as important as the anal-
gesic-induced interruption of reflex behaviors evoked by sensory

stimuli. It is therefore important to develop animal models to evaluate
the non-reflexive component in pain. We have used different beha-
vioural outcomes that depend on higher brain processing to measure
how different painful stimuli affect them and how they can be differ-
entially modulated by drugs. We conclude that the implementation of
non-reflexive outcomes during analgesic selection and profiling testing
will improve preclinical translation.
# S3: ISCHEMIA AND REPERFUSION

## S3-1
CEREBROVASCULAR PATHOLOGY: NOVEL PHARMACOLOGICAL STRATEGIES

Cuartero M.I., De la Parra J., Pérez-Ruiz A., Bravo-Ferrer I.,
Medina V., Hernández-Jiménez M., García-Segura J.M., Lizasoain I.,
Moro M.A.

Unidad de Investigación Neurovascular, Departamento de
Farmacología, Facultad de Medicina, Universidad Complutense de
Madrid, and Instituto de Investigación Sanitaria Hospital 12 de
Octubre (imas12), Madrid, España

Cerebrovascular injury caused by stroke is the major cause of neuro-
logical disability and the second cause of dementia. Specifically, the
prevalence of developing new dementia among first-ever stroke
patients is approximately 10% and the chance of developing delayed
post-stroke dementia is as high as 50% among stroke survivors. Stroke
is thus a major socio-economic problem for societies and health-care
systems world-wide. Despite this high disease burden, there is no
specific treatment for post-stroke dementia. One of the reasons for this
is that no suitable animal models are available that reliably allow for
the investigation of mechanisms of post-stroke cognitive deficits and
for the preclinical evaluation of candidate drugs. In our presentation,
we will describe an experimental paradigm in which some mice under-
going stroke show cognitive impairments in hippocampal-dependent
tests, and that may thus serve for the identification of individual mech-
anisms underlying cognitive manifestations of vascular cognitive
impairment. In this context, hippocampal neurogenesis, through which
new neurons integrate into hippocampal circuits driving memory pro-
cesses, has been stated to contribute to cognitive function. We will
report new evidence on the involvement of hippocampal neurogenesis
in cognitive impairment in the chronic phase of ischemic stroke. In
addition, we will discuss the role of Aryl hydrocarbon Receptor
(AhR), a bHLH/PAS ligand-activated transcription factor, as a putative
novel pharmacological target in this setting.

## S3-2
NOVEL THERAPIES FOR ACUTE ISCHEMIC STROKE:
FROM PHARMACOLOGICAL NEUROPROTECTION TO
VASCULOPROTECTION. THE URIC ACID EXPERIENCE

Delgado S.A.

Comprehensive Stroke Center at Hospital Clínic Barcelona, Barcelona,
Spain

Acute ischemic stroke is a major cause of morbidity and mortality. The most effective treatment is early reperfusion, although less than half of patients who are finally treated obtain permanent benefits. The combination of rapid reperfusion and neuroprotective therapies would be of added value for maximizing the beneficial effects of rapid reperfusion and to reduce the harmful consequences of reperfusion injury. Given the relevance of oxidative stress in the physiopathology of brain ischemia, the use of antioxidant molecules, such as uric acid, could translate into effective neuroprotective effects. Uric acid (UA) is the most potent natural antioxidant, and its exogenous administration in experimental models of brain ischemia is neuroprotective and has synergistic effects when administered alongside alteplase, a thrombolytic drug. Additionally, UA also attenuates vessel-wall changes induced by ischemia by reducing local oxidative stress and inflammation thus suggesting the relevance of additional vascular protection to direct effects on neurons to enhance post-stroke recovery. In humans, UA therapy is

safe and has measurable neuroprotective effects, especially in patients
with pre-treatment hyperglycemia or in women. Given the encouraging
preclinical and clinical data regarding the potential neuro/vasculopro-
tective effects of UA administration in combination with reperfusion in
acute brain ischemia, the development of adequately powered pivotal
confirmatory clinical trials are warranted.

## S3-3
OXYCYTE: AN INTRAVENOUS OXYGEN CARRIER
CAPABLE OF DIAGNOSING AND TREATING ACUTE
ISCHAEMIC STROKE

Macrae I.M.¹, Deuchar G.A.², Shirley R.¹, Roy L.¹, Brennan D.²,³,
Holmes W.M.¹, Gonzalez L.¹, McCabe C.¹, Muir K.W.³, Dani K.⁵,
Santosh C.²,³

¹Institute of Neuroscience & Psychology, College of Medicine,
Veterinary, and Life Sciences, University of Glasgow, Glasgow, UK;

²Aurum Biosciences, Glasgow, UK; ³Institute of Neurological
Sciences, Queen Elizabeth University Hospital, Glasgow, UK

Glasgow Oxygen Level Dependent (GOLD) technology is a novel
stroke management product in development by Aurum Biosciences,
which offers unique theranostic benefits through simultaneous diagnos-
tic and therapeutic applications. MRI-based techniques are combined
with an intravenous oxygen-carrying emulsion (Oxycyte®) and an oxy-
gen challenge (OC- increased inspired oxygen) to identify and support
the survival of ischaemic penumbra, injured but potentially salvageable
hypoperfused tissue.

Proof-of-concept for the diagnostic was established using 100% O₂
alone in rodent stroke models¹,² with T2*OC MRI translated to 3T
clinical scanners and tested in stroke patients³. Limitations identified
when using 100% O₂ were overcome by incorporating Oxycyte
nanoparticles (30–40 times smaller than red blood cells) injected i.v.
Oxycyte (4.5 ml/kg, i.v) combined with hyperoxia (40–50% O₂)
improves identification of T2*OC defined penumbra on MRI scans⁴
and improves sensitivity to detect changes in penumbra lactate levels.

Oxycyte + hyperoxia also improves oxygen transport through the ischaemic microcirculation via any remaining plasma flow, improving oxygen levels in penumbra and thereby limiting damage. Serial MRI scanning following stroke reveals brain damage is significantly reduced in rats treated with 3 ml/kg Oxycyte + hyperoxia compared to normoxic controls or hyperoxia alone. Oxycyte + hyperoxia reduces infarct size and improves functional outcome in rat stroke models (transient & permanent middle cerebral artery occlusion) and shows no contra-indications when combined with t-PA, MRI or CT contrast agents in a rat embolic stroke model.

**Conclusions:** GOLD shows potential to improve acute ischaemic stroke patient management: diagnostically providing a single stratified measure of tissue viability via MRI-based metabolic imaging while therapeutically supporting penumbra survival by improving oxygen delivery.

**References:**

1. Santosh C, et al. J Cereb Blood Flow Metab 2008;28:1742-53.

2. Holmes WM, et al. NMR Biomed 2012;25:295-304.

3. Dani KA, et al. Ann Neurol 2010;68(1):37-47.

4. Deuchar GA, et al. J Cereb Blood Flow Metab 2013;33:1422-8.

Oxycyte® provided by Tenax Therapeutics Inc. (Morrisville, NC,
USA).

**Key words:**

stroke; MRI; penumbra; rat.
S3-4
PROTECTION AGAINST MYOCARDIAL REPERFUSION
INJURY BY TARGETING MITOCHONDRIAL SUCCINATE
DEHYDROGENASE

Rodríguez-Sinovas A., Valls-Lacalle L., Barba I., Miró-Casas E.,
Ruiz-Meana M., García-Dorado D.

Cardiovascular Diseases Research Group, Vall d'Hebron Research
Institute, Barcelona, Spain

**Background:** Previous studies have demonstrated the importance of reactive oxygen species (ROS) generation during flow restoration after ischemia in reperfusion injury. In a recent study we analyzed the role played by reverse electron transfer from mitochondrial complex II to complex I in ROS generation during reperfusion. For this purpose, we reversibly inhibited succinate dehydrogenase with malonate, and analyzed its effects on infarct size.

**Methods and results:** Before testing the effects of succinate dehydro-
genase inhibition in reperfusion injury, we assessed malonate actions
in normoxic hearts. Isolated mice hearts were treated, under normoxic
conditions, with increasing concentrations of disodium malonate
(0.03–30 mM, *n* = 4). Malonate induced a concentration-dependent

decrease in left ventricular developed pressure (EC50 = 8.05 ± 2.11 mM). In isolated mice hearts submitted to 35 min of ischemia followed by reperfusion (60 min), malonate, given continuously from 15 min before ischemia to the end of reperfusion, at either 3 or 10 mM (n = 7–8/group) reduced infarct size, an effect associated with reduced LDH release and improved functional recovery during reperfusion. To assess its effects on reperfusion injury, malonate 3 mM was given only during the first 15 min of reperfusion. Under these conditions, malonate was still able to reduce infarct size (24.57 ± 2.32 vs. 39.84 ± 2.78 in controls, *p* = 0.001, n = 7–8/group) and LDH release (125.41 ± 16.82 vs. 189.20 ± 13.74 U/g dry tissue/15 min, *p* = 0.015) and to improve functional recovery at the end of reperfusion. Furthermore, malonate increased myocardial succinate content (¹H-NMR), reduced tissue ROS production (MitoSOX staining) during reperfusion, and reduced mitochondrial permeabilization (calcein retention). These data have been recently confirmed in an in situ pig model of transient coronary occlusion (infarct size of 59.62 ± 4.00% of area at risk in control animals vs. 36.46 ± 5.35% in animals treated with 10 mM malonate).

**Conclusion:** Succinate dehydrogenase inhibition with malonate at the onset of reperfusion reduces reperfusion injury in mice and pig hearts.
SYMPOSIA 4:G PROTEIN-COUPLED RECEPTORS
PHARMACOLOGY

S4-1
“THE DRUG INVENTION TEMPLATE: FROM “CRAFTED ROUND THE NATURAL HORMONE” TO “SCULPTED TO FIT THE RECEPTOR”

McGrath I.

University of Glasgow, Glasgow, UK

Over the last 60 years receptors for extracellular messengers have moved from being entirely theoretical concepts (black boxes) to molecules whose structure is being considered in 3D and time. In this introduction to the symposium on 7TM/GPC Receptors I will discuss how drug invention following the key and lock analogy (agonist and receptor) has changed. Initially we knew only about the key, with no knowledge of the lock, and modified the key in an attempt to disable the lock. Now that we have some knowledge of the structure of the lock, does that increase the efficiency of designing keys?

S4-2
NEW APPROACHES TO MODULATE G PROTEIN-COUPLED RECEPTORS

Pardo L.

Laboratori de Medicina Computacional, Unitat de Bioestadística,
Facultat de Medicina, Universitat Autònoma de Barcelona, Barcelona,
Spain

Despite the proven success of GPCRs as drug targets, useful ligands do not exist for the majority of them. The main reason being that the orthosteric binding sites across members of a GPCR subfamily for a particular endogenous ligand are often highly conserved, making it difficult to achieve high selectivity for specific GPCR subtypes. Thus, the emerging knowledge of the structure and physiological functions of GPCRs has begun to alter the approaches for drug discovery. These novel approaches to modulate GPCRs involve, in addition to traditional drug discovery programs dominated by efforts to develop compounds that act as agonists or inverse agonists by binding to the orthosteric site for endogenous hormones, the discovery of bitopic ligands that bind the orthosteric site as well as a less conserved site at the extracellular entrance, multivalent ligands that target physiologically relevant GPCR hetero-oligomers, or allosteric ligands that bind at allosteric sites and act in conjunction with the endogenous ligand. These allosteric ligands, which have been proven highly successful for ligand-gated ion channels, do not bind to the orthosteric ligand binding site but instead act at an alternatively located binding site (allosteric site) that is distinct from the orthosteric site. Allosteric modulators are categorized in negative (NAM) or positive (PAM) depending on their ability to decrease or increase the action (affinity and/or efficacy) of the orthosteric ligand.

In this presentation, we will explore new approaches to modulate GPCRs and the challenges associated with the design of new type of ligands.

S4-3
OPTOPHARMACOLOGY: LIGHTING UP G PROTEIN-COUPLED RECEPTORS

Fernández-Dueñas V.<sup>1,2</sup>, López-Cano M.<sup>1,2</sup>, Taura J.<sup>1,2</sup>, Ciruela F.<sup>1,2</sup>

¹Unitat de Farmacologia, Departament de Patologia i Terapèutica
Experimental, Facultat de Medicina i Ciències de la Salut, IDIBELL-
Universitat de Barcelona, Barcelona, Spain; ²Institut de Neurociències,
Universitat de Barcelona, Barcelona, Spain

G Protein-coupled receptors (GPCR) constitute the largest group of cell-surface receptors involved in signal transduction and the most important pharmaceutical drug targets. Numerous drugs targeting GPCR have been shown to exert beneficial therapeutic effects, but in many cases, they also provoke undesired adverse effects. Thus, a major aim in pharmacological research consists of developing novel drugs that may have effectiveness but with limited side effects. Optopharmacology, which is based on the use of light-activable compounds that can elicit their effect only upon light activation, is a promising approach. We have developed light-dependent GPCR (i.e. adenosine, glutamate receptors) ligands that allow photocontrolling, both *in vitro* and *in vivo*, receptors' activity. Interestingly, we challenged these light-activable compounds in heterologous systems, neuronal cultures and in mouse models, and demonstrated the ability of precisely, in time and space, deliver the active compound at the desired targets. Overall, optopharmacology may permit achieving better benefit/risk therapeutic balances when using GPCR-based drugs. This work was supported by grants MINECO/ISCIII (SAF2014-55700-P, PCIN-2013-019-C03-03 and PIE14/00034), the Catalan government (2014 SGR 1054), ICREA (ICREA Academia-2010) and Fundació la Marató de TV3 (Grant 20152031) and FWO (SBO-140028). We thank Esther Castaño and Benjamín Torrejón from the Scientific and Technical Services (SCT) group at the Bellvitge Campus of the University of Barcelona for their technical assistance.

S4-4
PROBING THE MOLECULAR MECHANISM BEHIND THE COGNITIVE IMPAIRMENT INDUCED BY THC

Botta J.¹, Cordomí A.², Bondar A.³, Lazar J.³, Pardo L.²,⁴,

McCormick P.J.¹,⁵

¹School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich, UK; ²Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain; ³School of Biological and Chemical Sciences, Queen Mary, University of London, London, UK; ⁴Institute of Nanobiology and Structural Biology of GCRC ASCR, Laboratory of Cell Biology, University of South Bohemia, Bohemia, Czech Republic; ⁵School of Veterinary Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, UK

There is high medical interest in harnessing the beneficial effects of delta-9-tetrahydrocannabinol (THC). We found that the serotonin 2AR receptor (5-HT2AR), a major target for anti-psychotics, is necessary for the amnesic- and anxiolytic-like effects of THC, but not for its hypolocomotor, hypothermic, anxiogenic and analgesic effects. While investigating the underlying molecular mechanism we discovered that CB1 cannabinoid receptor (CB1R) and 5-HT2AR form heteromers. Remarkably, we found that the formation of this heterocomplex leads to a switch in G-protein coupling for 5HT2AR from Gq to Gi proteins
and to a reduction in cell signaling. We demonstrated with behavioral experiments that via this heteromer complex, antagonists of 5-HT2AR can mitigate the amnesic and anxiolytic effects of THC. We then mapped which amino acids are responsible for these interactions and in so doing provide a framework for how receptor crosstalk can occur. These genetic, molecular and pharmacological data imply that the detrimental side effects of cannabis use can be avoided without

affecting its beneficial properties through a specific modulation of the CB1R-5-HT2AR heterocomplex. This potential for modulation, either in the form of disruption of the heteromer or its pharmacological blockade represent a new mechanism for both THC and potentially anti-psychotics and clearly indicates that CB1R-5-HT2AR heteromers are new targets for drug design.
# S5: CARDIOVASCULAR AND SMOOTH MUSCLE PHARMACOLOGY

## S5-1
NEW DRUGS IN THE TREATMENT OF ACUTE HEART FAILURE: HOPE OR HYPE?

Tamargo J.

Department of Pharmacology, School of Medicine, CIBERCV, Universidad Complutense, Madrid, Spain

Acute heart failure (AHF), defined by a rapid or gradual onset of signs and symptoms of HF requiring urgent therapy, is a heterogeneous, multifactorial and progressive syndrome presenting a wide spectrum of phenotypes often associated with multiple comorbidities. AHF represents a major health problem due to its high prevalence, morbidity, mortality and significant healthcare costs, and a therapeutic challenge for the clinician. In the last 25 years, multiple potential therapeutic targets involved in the genesis/progression of AHF have been identified and many promising new drugs were investigated. However, the treatment of AHF has not changed much over this time as new drugs improved signs and symptoms, but failed to improve HF outcomes when compared to placebo or conventional therapies. These considerations provide the stimulus for the development of new drugs that target the underlying pathophysiological processes leading to progressive myocardial dysfunction and unfavourable remodelling and improve long-term outcomes of patients with AHF. The development of new safer and more effective drugs for AHF should be based on well-supported hypotheses, robust preclinical data and well-designed randomized controlled trials. Interestingly, a repeated finding in AHF treatment has been the discrepancy between the positive results found in preclinical studies and the lack of efficacy and safety in phase II and III clinical trials. In this presentation the mechanisms of action, efficacy and safety of new drugs under development for the treatment of AHF (inotropes, vasodilators, hormones, atrial natriuretic peptides and NO-independent stimulators and activators of soluble guanylate cyclase, β-arrestin-biased AT1R ligands) and the possible explanations for the discrepancy between preclinical and phase II-III trials will be analyzed. A better understanding of AHF triggers and pathophysiology will allow to identify new targets and develop novel therapeutic approaches that might prevent the progression of myocardial dysfunction and improve outcomes.

## S5-2
NOVEL MEDIATORS OF INFLAMMATION IN VASCULAR DAMAGE IN HYPERTENSION

Briones A.M.<fnref n="1" />, García-Redondo A.B.<fnref n="1" />, Serrano L.<fnref n="1" />, Alonso M.J.², Salaices M.<fnref n="1" />

Hypertension is a global burden that significantly contributes to target organ damage and is a major risk factor for cardiovascular diseases. Despite this knowledge recent evidence suggest that over the past 25 years, the number of individuals with prehypertension or established hypertension has increased substantially and this is associated with the estimated number of deaths. Different factors and mechanisms are involved in hypertension development and maintenance but at vascular level specific alterations are found in both patients and hypertension models. In general, endothelial dysfunction, vascular remodeling

and increased vascular stiffness are key hallmarks of hypertensive vascular disease and they predict target organ damage and the development of future cardiovascular disease. Different factors and mechanisms have been involved in the vascular alterations observed in hypertension and it is now well accepted that low-grade inflammation triggered by several components of the innate and adaptive immune systems plays a key role in promoting a misbalance between vasoconstrictor/growth promoter and vasodilator/growth inhibitor factors in hypertension. Proinflammatory cytokines, oxidative stress derived from novel sources such as lysyl oxidase, prostanoids from the inducible isoform of the microsomal prostaglandin E synthase-1 and other mediators seem to have a role in vascular alterations in hypertension through their effects in NO availability, vascular smooth muscle cells proliferation or extracellular matrix deposition.

**Key words:** Hypertension; inflammation; endothelial dysfunction; vascular remodeling.

## S5-3
DRUGS FOR PULMONARY HYPERTENSION: OLD STORIES OF PARTIAL SUCCESS, PARTIAL FAILURE AND NEW PERSPECTIVES

Pérez-Vizcaíno F.

Universidad Complutense de Madrid (Spain) and Ciber Enfermedades Respiratorias, Madrid Spain

Pulmonary arterial hypertension is a rare life-threatening progressive disease characterized by vasoconstriction, vascular remodeling and thrombosis. Over the last 20 years we have witnessed the introduction of an unprecedented large number of drugs belonging to different classes which are directed mainly to reduce the vasoconstrictor component. They restore endothelial function by mimicking or inhibiting endothelial derived factors: NO (inhaled NO, phosphodiesterase 5 inhibitors and soluble guanylyl cyclase stimulators) prostacyclin (epoprostenol and its analogues), and endothelin-1 (endothelin-1 receptor antagonists). Unfortunately, there are no approved specific therapies for the most common "non-arterial" forms of PH: PH due left heart disease (group 2) and associated to lung diseases and hypoxia. Current therapies improve symptoms and prolong survival but the disease remains fatal. Vasodilators are limited by several factors. First, they lack pulmonary selectivity, thus their systemic effects leading to hypotension often preclude the use of effective doses. Second, despite most approved drugs show antiproliferative effects to some extent, aggressive proliferative phenotypes are often drug-resistant. However, attempts to directly address proliferation with kinase inhibitors have failed in clinical trials. Third, vasodilators are often unable to induce effective vasodilation depending on the underlying vasoconstrictor mechanism. Finally, they may uncouple ventilation-perfusion ratio. Hypoxic pulmonary vasoconstriction (HPV) represents a crucial protective mechanism that redistributes blood flow away from diseased (hypoxic) lung tissue to the best oxygenated alveoli at the expense of elevated pulmonary pressure. Vasodilators, by inhibiting HPV, may also increase blood flow to poorly-ventilated areas of the lung decreasing arterial oxygenation.

<footnote n="1">¹Department of Pharmacology, Faculty of Medicine, Universidad Autónoma de Madrid, Madrid, Spain; ²Department of Ciencias Básicas de la Salud, Universidad Rey Juan Carlos, Alcorcón, Spain</footnote>
S5-4
PLEIOTROPIC ACTIONS OF LOSARTAN METABOLITES IN HYPERTENSION

Zalba G.

Department of Biochemistry and Genetics, University of Navarra,
Pamplona, Spain

Losartan is an antihypertensive drug that exerts its effects by the blocking of the angiotensin II type 1 (AT₁) receptor, which also exhibits a variety of pleiotropic effects. Losartan is a prodrug metabolized by the cytochrome-P-450 pathway in the liver producing 2 metabolites, EXP3174 and EXP3179. EXP3174 is the final metabolite and the pharmacological blocker of the AT₁ receptor by which losartan exerts its antihypertensive actions. EXP3179 is an aldehyde intermediate metabolite that has no AT₁ receptor blocking properties and mediates a variety of AT₁ receptor-independent, non-hemodynamics actions. Thus EXP3179 metabolite (i) mediates the anti-inflammatory properties of losartan by abolishing cyclooxygenase-2 upregulation, (ii) stimulates endothelial NO synthase and suppresses tumor necrosis factor-α-

induced apoptosis, (iii) acts as a peroxisome proliferator-activated receptor-γ-agonist, (iv) inhibits collagen-dependent platelet activation, and (v) blocks the NADPH oxidase in phagocytic and vascular cells, an effect that appears to depend on a protein kinase C-dependent mechanism. Traditionally, losartan has been demonstrated to reduce myocardial fibrosis and left ventricular stiffness in hypertensive patients, effects associated with the reduction of the content of myocardial collagen fibers. Several recent studies have underlined the key role that the degree of collagen-cross linking (CCL) within the collagen fibers, a process catalyzed by the enzyme lysyl oxidase (LOX), plays on cardiac stiffness and resistance to fibers degradation. In a model of experimental hypertension, rats treated with N>G-nitro-L-arginine methyl ester, EXP3179 metabolite prevents LOX, CCL increase, as well as fibrosis. This effect is obtained without normalization of blood pressure. In contrast, 3174 metabolite prevents blood pressure increase and attenuates fibrosis but do not modify LOX and CCL. Thus, and despite a lower antihypertensive effect, EXP3179 shows higher anti-fibrotic efficacy than EXP3174.

**Key words:** Losartan; Hypertension; Nitric oxide; Myocardial fibrosis.
# S6: NATURAL PRODUCTS IN PHARMACOLOGY

## S6-1
QUALITY OF HERBAL MEDICINAL PRODUCTS: CONTRIBUTION OF THE EUROPEAN PHARMACOPOEIA

Cañigueral S., Vila R.

Unitat de Farmacologia, Farmacognòsia i Terapèutica, Facultat de Farmàcia i Ciències de l'Alimentació, Universitat de Barcelona, Barcelona, Spain

Herbal medicinal products (HMP) and Traditional HMP contain herbal drugs (HD) or herbal preparations (HP) (extracts, essential oils, etc.) as active pharmaceutical ingredients. The quality of HMP is a challenge, mainly due to the difficulties linked to the variability and complexity of HD and HP, the limited knowledge of their active constituents, the possible adulterations and contaminations, as well as the influence of the production processes. Overcoming this challenge requires to have the quality as an objective from the beginning of the production chain, to establish strict controls on all stages, to improve the knowledge on the product, to optimize and validate the production processes, to work under accepted international guidelines and to establish quality specifications (¹,²).

In setting quality specifications, the European Pharmacopoeia (Ph.Eur.), an organization in the European Directorate for the Quality of Medicines and Health Care (EDQM) of the Council of Europe, is a key tool for maintaining the high levels of quality of HMP in Europe. This is because the Ph. Eur. is an updated and transparent regulatory document, harmonized and agreed by 38 countries, which provides a unique collection of methods of analysis and ca. 330 monographs of HD and HP (²,³); plant parts (61%), extracts (14%), essential oils (10%), exudates, starches, gums and mucilages (8%) and fatty oils and waxes (7%). Three permanent groups (13A, 13B and 13H) and several working parties (TCM, Extracts, Pesticides, etc.), comprising more than 70 experts of 21 countries, from industry or private sector (46%), universities or public research centres (31%) and medicines agencies (23%), together with the staff of the EDQM, make possible these achievements.

**References:**

1. Cañigueral S, et al. Pharm Med 2008; 22(2):107-118.

2. Cañigueral S. Revista de Fitoterapia 2013; 13(2): 101-122.

3. EDQM. European Pharmacopoeia, 9th Edition, supplement 9.2. Council of Europe, Strasbourg (2017).

## S6-2
FLAVONOIDS IN THE TREATMENT OF MENTAL DISORDERS

de la Torre R.

Hospital del Mar Medical Research Institute (IMIM), Barcelona, Spain

Several mental disorders like dementia and age-associated cognitive decline could be preventable depending on different aspects of life-style, in particular exercise and diet, that may affect neural function and consequent cognitive performance throughout the life course. A balanced diet and regular exercise can protect the brain and ward off mental disorders. Among minor dietary components, flavonoids, found in a variety of fruits and vegetables, are a group of promising bioactive compounds capable of influencing different aspects of brain function, including cerebrovascular blood flow and synaptic plasticity. Flavonoids health benefits can be considered in the context of diet or isolated from food matrix and used at doses higher than those provided by diet in the search of targeted pharmacological effects. This presentation will focus on the use of flavonoids as nutraceuticals.

Flavonoids can exert direct vs. indirect effects via different mechanisms of action. They are known to interact directly with neuronal receptors (i.e. estrogenic or glutamate) and kinase signaling pathways (i.e. DYRK1A) which are key to neuronal activation and communication and synaptic strengthening. Alternatively or concurrently, flavonoids can positively affect peripheral and cerebrovascular blood flow, which may be an indirect effective mechanism impacting brain health and cognition. Examples will be provided of the therapeutic potential of flavonoids for the improvement of cognitive function and adaptive functionality in intellectual disabilities like Down and Fragile X syndromes (epigallocatechin gallate), the treatment of substance abuse disorders like the fetal alcohol spectrum disorders (epigallocatechin gallate) or cocaine addiction (isoflavones) and the deceleration of cognitive decline (flavanones).

## S6-3
MARINE DERIVED MEDICINES FOR THE TREATMENT OF CANCER: THE EXPERIENCE OF PHARMAMAR

Fudio S.

PharmaMar SA, Colmenar Viejo, Madrid, Spain

Marine biodiversity is almost unlimited and yet to be broadly discovered. Marine exploration for pharmaceutical purposes is quite recent (<30 years old) and there are already some successful results, such as in the field of oncology. In this regard, when a tumor screening hit is detected on crude extracts from marine organisms, chromatographic purification of active samples is carried out to attempt isolation of a new chemical entity. This natural candidate may not be necessarily a drug-like molecule. While a cost-effective route of supplying the molecule needs to be made available, it should have the potential to reach the site-of-action in man, at the required concentration, for the necessary duration and with an adequate safety window. Therefore, structural modification and/or drug delivery technologies may be put in place, along with the characterization of the adequate dose, regimen and route of administration in the clinical setting. Finally, a positive balance of benefits over risks in a specific unmet medical need should be considered of clinical relevance in order to apply for marketing approval.

During the last 30 years, PharmaMar has been focused in the search of oncology therapies from macro- and micro-organisms of marine origin and, as a result, has developed the first marine-derived compound to reach the oncology market.

## S6-4
DEVELOPMENT OF NEUROPROTECTIVE THERAPIES WITH PHYTOCANNABINOIDS FOR NEURODEGENERATIVE DISORDERS

Fernández-Ruiz J.¹,²,³

¹Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Investigación en Neuroquímica, Universidad Complutense, Madrid, Spain; ²Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain; ³Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain

Phytocannabinoids are compounds derived from cannabis plant, which, together with endogenous signalling lipids and synthetic derivatives, form a singular family of pleiotropic compounds active in the CNS,
but also in the periphery, and having promising therapeutic applica-
tions, including cytoprotection in neurodegenerative disorders. Such
application derives from their capability to preserve, rescue, repair and/
or replace neurons, astrocytes, oligodendrocytes and their precursor
cells against a myriad of insults that deteriorate the homeostasis and
integrity of these neural cells. These cytoprotective effects are facili-
tated by the location of specific targets for the action of phytocannabi-
noids (e.g. CB₁ and CB₂ receptors, endocannabinoid inactivating
enzymes, non-endocannabinoid targets) in key cellular substrates (e.g.
neurons, astrocytes, resting and reactive microglia, perivascular micro-
glial cells, oligodendrocytes and oligodendrocyte precursor cells, and
neural progenitor cells) and structures (e.g. blood-brain barrier [BBB]).
Such cellular locations enable phytocannabinoids to exert selective
control over the specific functions fulfilled by these cells in degenera-
tion, protection, and/or repair. For example, in the case of CB₁ recep-
tors, their activation inhibits glutamate release limiting excitotoxic
damage, enhances metabolic support exerted by astrocytes for neurons,
and promotes remyelination by increasing oligodendrocyte precursor
cell maturation. In the BBB, CB₁ receptor activation improves vascular

supply and limits peripheral cell infiltration. In the case of CB₂ recep-
tors located in astrocytes and reactive microglia, their activation
reduces the generation of fractalkine, TNF-α, IL-1β and other proin-
flammatory mediators by these activated glial cells. FAAH and MAGL
enzymes, and GPR55 and PPAR receptors have been also identified in
key cellular substrates being also proposed to mediate the cytoprotec-
tive activity of phytocannabinoids. In summary, the multiplicity of
active targets for phytocannabinoids places this family of natural com-
pounds in a promising position for developing novel cytoprotective
therapies using a broad-spectrum phytocannabinoid or combinations of
compounds, a circumstance essential in neurodegenerative disorders, in
which neuronal damage is the result of a concerted action of different
neurotoxic processes (e.g. excitotoxicity, oxidative stress, protein
aggregation, mitochondrial failure, glial reactivity, inflammation)
demanding a multi-target strategy capable to limit all these processes
at the same time.

Supported by MINECO (SAF2015-68580-C2-1-R) and CIBERNED
(CB06/05/0089).
# S7: BEING IN THE RIGHT PLACE IN THE RIGHT TIME: TARGETING OXIDATIVE STRESS

## S7-1
MEASURING LIGAND CONCENTRATION WHERE IT MATTERS: ASSESSING THE “MICRO PK/PD” OF ADENOSINE A2A RECEPTOR LIGANDS

Lochray J., Gherbi K., Sykes D.A., Briddon S., Charlton S.J.

School of Life Sciences, University of Nottingham, UK

We have recently demonstrated that, dependent upon their physio-
chemical properties, β₂-adrenoceptor ligands can interact directly with
phospholipids, increasing their local concentration and directly influ-
encing the measured association rate constant at the receptor (Sykes
et al, 2014). The aim of this current study was to expand upon this
observation by directly measuring the local ligand concentration and
binding kinetics of two fluorescent adenosine A2a receptor ligands
with identical pharmacophores (xanthine amine congener [XAC]) and
fluorophores (BODIPY 630/650-X-SE), but varying linker regions to
modulate the physicochemical properties. The first of these two
ligands, XAC-X-BY630, is more lipophilic than the second,
CA600245.

The local aqueous concentration of each ligand was measured using
fluorescence correlation spectroscopy (FCS) in a detection volume of
~0.25 fL at distances 2–200 µm above live CHO cells. Ligands were
added at 10-fold below their K<sub>D</sub> concentrations (XAC-X-BY630 at
5 nM, CA600245 at 15 nM). The binding kinetics of the two ligands
was assessed by measuring the time-resolved fluorescence energy
transfer (TR-FRET) between the terbium-labelled A2a receptor and
fluorescent ligand over time. The aqueous concentration of both
ligands was dramatically higher in the local vicinity of the plasma
membrane (2 µm above) than in bulk aqueous phase (200 µm
above). For example, at 3 µm above the membrane, the measured
concentration of CA600245 was 83.8 ± 23.1 nM (n = 6), whilst the
more lipophilic XAC-X-BY630 was even higher at
656.1 ± 212.7 nM (n = 6). This increased local concentration influ-
enced the association kinetics of the XAC derivatives with XAC-X-
BY630 having a significantly faster on-rate (1450 ± 450/mM/min;
n = 4) than CA600245 (462 ± 109/mM/min; n = 4) (p < 0.05,
unpaired t-test).

These data demonstrate that lipophilicity can increase the local concen-
tration of drug immediately above the cell membrane, potentially dis-
torting the measured pharmacological values. We propose it is
critically important to consider this “micro PK-PD” when determining
the pharmacology of novel receptor ligands.

Sykes et al (2014) *Mol Pharmacol* 85(4):608–17

## S7-2
MOLECULAR AND FUNCTIONAL IMPACT OF OXIDATIVE STRESS IN COPD

Kirkham P.

Department of Biomedical Sciences & Physiology, University of
Wolverhampton, Wolverhampton, UK

Oxidative stress is plays a major pathophysiological role in many lung
diseases, and is considered to be the principle predisposing factor in
the pathogenesis of COPD. Oxidative stress can cause alterations in
cellular redox status, activate redox sensitive kinase signaling pathways
and transcription factors. Oxidative stress can also impact on chromatin
modification enzymes resulting in reduced corticosteroid efficacy, as

well as triggering epigenetic changes that lead to increased pro-inflam-
matory responses. Some of the manifestations of oxidative stress arise
as a result of tissue damage by free radicals, through lipid peroxidation
for example, forming reactive carbonyls or carbonyl stress. This in turn
can lead to protein modification which can affect protein and cellular
function, along with wider systemic impacts that may lead to associ-
ated co-morbidities with COPD. This presentation will highlight how
oxidative stress arises in the lung and leads to increased carbonyl
stress, and how this impacts on inflammation and autoimmunity and
the development of emphysema and small airway disease in COPD.
Finally, a number of studies have shown that there is no single "magic
bullet" to combat oxidative stress, but instead a combination therapy,
targeting oxidative stress in the various sub-cellular compartments,
may prove to be more effective in COPD, with several promising
approaches on the horizon.

**Key words:** Oxidative stress; COPD; Inflammation; autoimmunity.

S7-3
TARGETING MITOCHONDRIA IN OXIDATIVE STRESS BY
MODULATION OF POTASSIUM CHANNELS

Dolga A.

University of Groningen, Groningen, the Netherlands

Alteration of mitochondrial metabolism and calcium homeostasis is highly associated with the pathology of neurodegenerative diseases. Small conductance calcium-activated potassium (SK) channels provide protection in different paradigms of neuronal cell death. Recently, these channels were identified at the inner mitochondrial membrane, however, their particular role in the observed neuroprotection remains unclear. In this study we aim to investigate the distinct role of SK channels in protection against glutamate toxicity, and to identify a potential role of SK channels in the regulation of mitochondrial metabolism and calcium uptake, two key players in neuronal cell death. Our results demonstrate that overexpression of mitochondria-targeted SK2 channels enhanced CyPPA-mediated mitochondrial resilience by reducing mitochondrial ROS formation. These effects were inhibited by overexpression of a mitochondria-targeted dominant-negative SK2 channel mutant. Real-time analysis of neuronal cells revealed that SK2 channel activation, or enrichment with mitochondrial SK2 channels, attenuated mitochondrial respiration and mitochondrial calcium uptake. These findings strongly suggest that mitochondrial SK2 channels provide neuroprotection by regulation of the mitochondrial metabolism and mitochondrial calcium homeostasis in conditions where sustained mitochondrial damage determines progressive neuronal death.

S7-4
MITOCHONDRIAL DYSFUNCTION IS A KEY DRIVER OF
FIBROBLAST SENESCENCE. SO WHAT DO WE TARGET?

Knight D.

Head of School, Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, Australia

**Introduction and Rationale:** The limited success in identifying and targeting important drivers of idiopathic pulmonary fibrosis (IPF) is likely because of the disease's complexity and heterogeneity. Previous studies have compared gene or protein expression from biopsies or
random samples of IPF lung to normal or disease controls. However, regional variation and typical progression of IPF provides a unique opportunity to investigate disease pathogenesis.

**Hypothesis:** Region-specific cues from the microenvironment are central to the pathogenesis of IPF.

**Methods:** We are integrating multiple samples isolated from the same individual lung; including pan-omics based profiling and imaging with cell-based studies with the severity of pathology in human disease, in order to comprehensively map the dynamic processes that regulate current fibrosis and predict future disease.

**Results:** Preliminary MALDI/MS imaging identified the spatial distribution of 198 protein signals from different non-fibrotic lung sections and 163 protein signals from the corresponding IPF sections, including immunoglobulins and ECM fragments. Cell culture data suggest that fibroblasts also show region-specific profiles of senescence, ECM production and proliferation.

**Conclusions:** The use of an individual lung for systems-based approaches offers unique insight into the pathogenesis of IPF, with the added advantage of minimizing disease co-morbidities and genetic heterogeneity.

**Key words:** idiopathic pulmonary fibrosis (IPF), lung disease.
# S8: INFLAMMATION

## S8-1
L-SELECTIN SHEDDING IS THE “GO” SIGNAL FOR NEUTROPHIL EFFECTOR FUNCTION

Davies J.¹, Rahman I.¹, Pombo J.¹, Petri B.², Zemp F.², Charras G.³, Kubes P.², Ivetic A.¹

¹Cardiovascular Division, BHF Centre of Research Excellence, King's College London, UK; ²Snyder Institute for Chronic Diseases, University of Calgary, Calgary, AB, Canada; ³London Centre for Nanotechnology, University College London, London, UK

We have reported that ectodomain shedding of L-selectin in monocytes occurs specifically during transendothelial migration (TEM) and not before (1). L-selectin shedding is critical in regulating front-back polarity of monocytes entering the subendothelial space, which in turn facilitates interstitial chemotaxis. We have recently extended our focus towards neutrophils and find that L-selectin shedding is similarly regulated during TEM (unpublished). In vivo, neutrophils are typically challenged with conflicting gradients of intermediate chemokines (e.g. IL-8) and end stage chemoattractants (e.g. mitochondrial fMLF) and are therefore required to prioritise their responses to such gradients. In this context, we explored the in vivo importance of L-selectin shedding in mediating neutrophil chemotaxis towards different ranking chemoattractants. By challenging the cremasteric muscle: first with chemokine (MIP-2/KC) and then with superfused fMLF (a damage/pathogen associated molecular pattern – DAMP/PAMP), or laser injury (to promote neutrophil swarming), we noticed that shedding of L-selectin was only ever apparent in neutrophils responding to DAMPs but not chemokines. Modelling these inflammatory responses in vitro revealed that human neutrophil chemotaxis through 3D collagen scaffolds towards fMLF gradients, but not gradients of the chemokine IL-8, was significantly attenuated when L-selectin shedding was genetically or pharmacologically blocked. Furthermore, release of superoxide in response to fMLF stimulation or phagocytosis of opsonised zymosan particles were also significantly attenuated when L-selectin shedding was blocked. Taken together, we conclude that L-selectin shedding is the “go” signal for neutrophil chemotaxis towards end-stage chemoattractants (e.g. fMLF) and effector function (e.g. phagocytosis and superoxide production). Future work will explore whether blocking L-selectin shedding is a suitable therapeutic target for minimising unwanted neutrophil effector function in the setting of acute sterile injury, such as myocardial infarction.

(1) L-selectin shedding is activated specifically within transmigrating pseudopods of monocytes to regulate cell polarity in vitro. Rzeniewicz K, Newe A, Rey Gallardo A, Davies J, Holt MR, Patel A, Charras GT, Stramer B, Molenaar C, Tedder TF, Parsons M, Ivetic A. Proc Natl Acad Sci U S A. 2015 Mar 24;112(12):E1461-70

## S8-2
ADIPOCYTOKINES: NOVEL THERAPEUTIC TARGETS IN ENDOTHELIAL DYSFUNCTION AND VASCULAR INFLAMMATION?

Peiró C.

Department of Pharmacology, Universidad Autónoma de Madrid, Madrid, Spain

The adipose tissue is not simply a storage site of fat mass, but also an organ that secretes a heterogeneous group of substances, the so-called adipocytokines, which may exhibit paracrine or endocrine actions. Adipocytokines range from classic cytokines, such as interleukin (IL)-1β or tumour necrosis factor-α, to vasoactive peptides or metabolic

regulators, such as leptin. They can be released not only by the visceral or subcutaneous adipose tissue, but also by epicardial or perivascular fat depots. In certain conditions, such as type 2 diabetes and obesity, an imbalanced production and release of adipocytokines has been proposed to negatively impact on vascular homeostasis, thus contributing to the development of vascular disease. In this context, adipocytokines have gained interest as potential pharmacological targets to retard or attenuate vascular complications associated to metabolic disorders. In recent years, we have explored the capacity of selected adipocytokines, such as IL-1β, visfatin/Nampt or soluble dipeptidyl peptidase-4 (sDPP4), to directly promote features of vascular damage, including vascular cell inflammation, endothelial dysfunction, impaired vascular reactivity or premature cell senescence, as well as the respective mechanisms of action involved. Moreover, we have observed that the pro-inflammatory signalling triggered by IL-1β in human vascular cells is exaggerated by the presence of extracellular glucose levels. This effect seems to rely on a higher uptake of glucose favoured by the adipocytokine. Once inside the cell, part of the excess glucose is diverted via the pentose phosphate pathway, which derives from the over-production of oxygen reactive species resulting in an over-activation of pro-inflammatory signalling. The deleterious vascular effects exerted by different adipokines can be pharmacologically blocked *in vitro* or *in vivo* by drugs that are either under development or already available in clinical practice for non-vascular indications. This opens new therapeutic horizons for the treatment of cardiometabolic complications.

Keywords: vascular, inflammation, endothelial dysfunction, adipocytokines

## S8-3
CXCL16/CXCR6 AXIS BLOCKADE IMPAIRS METABOLIC SYNDROME-ASSOCIATED ENDOTHELIAL DYSFUNCTION AND ABDOMINAL AORTIC ANEURYSM (AAA) DEVELOPMENT

Collado A.¹,², Marques P.¹,², Escudero P.¹,², Rius C.¹,², Martinez-Hervás S.²,³, Real J.T.²,³, Ascaso J.F.²,³, Piqueras L.², Sanz M.-J.¹,²

¹Department of Pharmacology, Faculty of Medicine, University of Valencia, Valencia, Spain; ²Institute of Health Research INCLIVA, Valencia, Spain; ³Endocrinology and Nutrition Unit, University Clinic Hospital of Valencia, Valencia, Spain

**Introduction and Objectives:** Clinical evidence indicates that metabolic syndrome (MS) is a precursor of endothelial dysfunction and an independent predictor of abdominal aortic aneurism (AAA). Since in both pathologies angiotensin-II (Ang-II) is a relevant player, the functional role of CXCL16/CXCR6 axis in MS, AAA formation and the mechanisms involved in Ang-II-induced CXCL16 arterial expression were investigated.

**Methods and results:** Flow cytometry revealed that MS patients showed increased platelet activation and percentages of circulating CXCR6-expressing platelets, CXCR6-expressing platelet-bound neutrophils, monocytes and CD8⁺ lymphocytes compared with aged-matched controls leading to enhanced CXCR6/CXCL16-dependent adhesion to the dysfunctional (Ang-II- and TNFα-stimulated) arterial endothelium determined by the dynamic model of the flow chamber. Endothelial Ang-II stimulation (1 µM) for 24 h caused significant CXCL16 up-regulation but its neutralization only significantly inhibited (49%) mononuclear leukocyte-adhesion to cells from arterial but not from venous origin. This response was found to be dependent on Nox5 expression and subsequent RhoA/p38-MAPK/NFκB activation. Ang-II-induced AAA formation in apolipoprotein E-deficient mice
(apoE<sup>-/</sup>, 1000 ng/kg/min, 28 days) showed AAA higher incidence, increased macrophage, CD3<sup>+</sup> and CXCR6<sup>+</sup> cell infiltration and enhanced neovascularization than unchallenged animals. These effects were accompanied by increased CCL2, CXCL16, CXCR6 and VEGF mRNA expression within the lesion and enhanced levels of circulating soluble CXCL16. These parameters were reduced in AT<sub>1</sub> receptor antagonists-treated animals (losartan, 30 mg/kg/day). Similar reductions in these parameters were found in apoE<sup>-/</sup> mice lacking functional CXCR6 receptor (CXCR6<sup>GFP/GFP</sup>). Therefore, CXCR6 expression on platelet-bound monocytes and CD8<sup>+</sup> lymphocytes may constitute a new membrane-associated biomarker for adverse cardiovascular events and pharmacological modulation of this axis may positively affect cardiovascular outcome in metabolic disorders linked to rennin-angiotensin system activation.

This study was supported by grants SAF2011-23777, SAF2014-57845R and PI15/00082 from the Spanish Ministry of Economy and Competiveness, Carlos III Health Institute, the European Regional Development Fund.

S8-4

INCREASED PLATELET AND T CELL ACTIVATION IN PATIENTS WITH PRIMARY HYPERCHOLESTEROLEMIA IS ASSOCIATED TO ENHANCED LEUKOCYTE-ARTERIAL ADHESION

Collado A.¹, Marques P.¹,², Perelló E.²,³, Martínez-Hervás S.²,³, Piqueras L.², Real J.T.²,³, Ascaso J.F.²,³, Sanz M.J.¹,²

¹Department of Pharmacology, Faculty of Medicine, University of Valencia, Valencia, Spain; ²Institute of Health Research INCLIVA, University Clinic Hospital of Valencia, Valencia, Spain; ³Department of Medicine, University Clinic Hospital-CIBERDEM, University of Valencia, Valencia, Spain

**Introduction and Objectives:** Primary hypercholesterolemia (PH) is associated with a marked increase of low-density lipoprotein cholesterol (LDL-C) which has been linked with augmented risk of premature cardiovascular disease (CVD). A low grade of systemic inflammation is often associated with PH and systemic inflammation is the main driver of premature atherosclerosis development. However, the systemic inflammatory response in PH has been poorly investigated. Therefore, we have determined the activation state of platelets and different T lymphocyte subsets and their atherogenic consequences.

**Materials and Methods:** Whole blood from 22 PH patients and 20 age-matched controls was analysed by parallel-plate flow chamber assay to evaluate platelet-leukocyte and leukocyte adhesion to TNFα-stimulated arterial endothelium. Flow cytometry was employed to measure platelet activation (P-selectin expression and circulating PAC-1<sup>+</sup> platelets). CD69 expression, a marker of leukocyte activation, was also determined in different lymphocyte subsets.

**Results:** Parallel-plate flow chamber revealed enhanced platelet-leukocyte and leukocyte adhesiveness to TNFα-stimulated arterial endothelial cells in PH patients vs. age-matched controls. PH patients presented greater numbers of activated circulating platelets (PAC-1<sup>+</sup>) with increased P-selectin expression than age-matched controls. Additionally, PH patients showed augmented numbers of platelet-lymphocyte aggregates than age-matched control subjects in different lymphocytes subsets (CD3, CD8, Th1, Th2 and Th17). Similarly, the activation state of CD4 lymphocytes such as Th1, Th2 and Th17 as

well as CD8<sup>+</sup> cells was significantly greater in PH patients than in age-matched controls.

**Conclusions:** We have provided evidence that increased platelet and lymphocyte activation in PH patients leads to enhanced arterial leukocyte adhesiveness. Therefore, since platelet/leukocyte- and leukocyte-arterial adhesion precedes atherogenic process development, it is plausible that the activation state of these immune players may predict the likelihood of suffering further cardiovascular events in PH patients.

S8-5

DPP4 DELETION IN ADIPOSE TISSUE PROMOTES HEPATIC INSULIN SENSITIVITY VIA IGF1

Romacho T.¹, Sell H.¹,²,³, Indrakusuma I.¹, Roehrborn D.¹, Jelenik T.¹,²,³, Castañeda T.R.⁴, Hartwig S.³, Weiß J.⁴, Al-Hasani H.⁴, Roden M.²,³,⁵, Eckel J.¹,³

¹Paul-Langerhans-Group for Integrative Physiology, Heinrich Heine University Düsseldorf, Düsseldorf, Germany; ²Institute for Clinical Diabetology, Heinrich Heine University Düsseldorf, Düsseldorf, Germany; ³German Center for Diabetes Research (DZD), München-Neuherberg, Germany; ⁴Institute for Clinical Biochemistry and Pathobiochemistry, Leibniz Center for Diabetes Research at Heinrich Heine University Düsseldorf, Düsseldorf, Germany; ⁵Department of Endocrinology and Diabetology, Medical Faculty, Heinrich-Heine University Düsseldorf, Düsseldorf, Germany

Besides a therapeutic target, dipeptidyl peptidase 4 (DPP4) is an adipokine secreted by adipose tissue (AT) whose serum levels are enhanced in obese and insulin-resistant patients. DPP4 might represent a missing link between increased AT mass and obesity-associated metabolic diseases. To explore the role of AT-derived DPP4 in diet-induced obesity, we generated an AT-specific DPP4 knockout (AT-DPP4-KO) mouse. Under high fat diet (HFD), circulating DPP4 levels (340 ± 32 ng/ml vs. 487 ± 30 ng/ml; *p* < 0.01) were reduced in KO mice, indicating AT as a relevant source of soluble DPP4. However, fasting and glucose stimulated serum concentrations of glucagon-like peptide-1 and gastric inhibitory peptide were similar in wild type and AT-DPP4-KO animals on HFD. KO animals under HFD showed improved oral glucose tolerance and suppression of endogenous glucose production during hyperinsulinaemic-euglycemic clamps. Although AT-DPP4-KO under HFD displayed increased body weight and fat mass, a beneficial remodeling was observed in a AT consisting of smaller adipocytes, enhanced M2 macrophage markers and decreased fibrosis markers. Upon HFD, knockout mice had lower IGF binding protein 3 (IGFBP3) levels in both AT and serum, while serum concentrations of free IGF1 were increased. Accordingly, exposure of human hepatocytes to recombinant IGFBP3 resulted in impaired insulin signaling and insulin-induced suppression of glucose production. Interestingly, type 2 diabetes patients treated with the DPP4 inhibitor sitagliptin, displayed increased serum free IGF1 and free IGF1/total IGF1 ratio. In conclusion, our study highlights a key role of adipose DPP4 in obesity-related metabolic disorders. Adipose DPP4 deletion under HFD triggers increased body weight followed by beneficial AT remodeling. In this scenario, adipose IGFBP3 production is reduced, leading to increased free IGF1 promoting hepatic insulin sensitivity. In summary, specific DPP4 deletion or inhibition arises as a novel therapeutic strategy in obesity to increase IGF1 and thus preserve hepatic insulin sensitivity.

**Keywords:** DPP4; adipokine; obesity; hepatic insulin sensitivity
# S9: PSYCHOPHARMACOLOGY

## S9-1
THE PROLYL OLIGOPEPTIDASE INHIBITOR IPR-19 AMELIORATES COGNITIVE DEFICITS IN MOUSE MODELS OF SCHIZOPHRENIA

Prades R.

Iproteos SL, Barcelona, Spain

Cognitive decline associated with schizophrenia is considered a key feature of the disease since it might precede the onset of the disease and continues after psychosis, being a predictor of the disorder evolution. Current antipsychotic drugs for positive or negative symptoms have little or no effect on cognitive deficits. Furthermore, an exacerbation of cognitive deficits by some antipsychotic drugs is plausible. Prolyl oligopeptidase (POP) is an 81-kDa monomeric serine protease that is expressed in brain and other tissues. Recent advances suggested that this enzyme participates in protein-protein interactions that control brain functions and cognitive processes. POP inhibition shows neuroprotective, anti-amnesic and cognition-enhancing properties in some rodent models. Here, we evaluated for the first time the potential of this target for the treatment of cognitive impairment associated with schizophrenia (CIAS). IPR-19, a potent and selective POP inhibitor, was tested in three cognition impairment mouse models associated with schizophrenia based on pharmacological subchronic phencyclidine and acute dizocilpine administration, and offspring from mothers with immune reaction induced by polyinosinic: polycytidylic acid administration during pregnancy. Acute IPR-19 administration (5 mg/kg, ip) was able to reverse the cognitive performance deficits of the three mouse models in the novel object recognition, T-maze and eight-arm radial maze tests. Although is not considered a behavioral test, IPR-19 also had positive effects on the prepulse inhibition test. The mode of action of IPR-19 differs greatly from other classical mechanisms unsuccessfully explored for the treatment of CIAS. The findings presented here become particularly valuable as a novel pharmacological mechanism option for the treatment of cognitive dysfunctions. Taking into account that CIAS represents the most treatment-elusive symptoms of schizophrenia, POP inhibition opens avenues for the treatment of this unmet clinical need.

## S9-2
ROLE OF SEROTONIN 5-HT3 RECEPTORS IN THE TREATMENT OF DEPRESSIVE DISORDERS

Callado L.F.

Department of Pharmacology, University of the Basque Country, UPV/EHU, Leioa and Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Spain

Depression is a chronic, recurring and potentially life-threatening mental illness with estimated lifetime prevalence up to 16% of the population. Despite current antidepressant treatments are generally safe and effective, less than a half of patients reach full remission. Therefore there is still a great need for faster acting, safer and more effective pharmacological treatments for depression. During the last decades, the monoamine hypothesis of depression has centre the development of new antidepressants. Thus, the increase of serotonin (5-HT) and/or noradrenaline (NA) availability in the synapse by the inhibition of their respective reuptake transporters underlies the therapeutic activity of the most widely prescribed antidepressant drugs. In this context, the 5-HT3 receptors are located postsynaptically on GABAergic interneurons in the brain stem regulating several neurotransmission systems. The activation of 5-HT3 receptors induces an inhibition of the release of 5-

HT and NA. Therefore, it was proposed that the antagonism of these receptors would lead to an increase in the release of monoamines that could improve symptoms of depression. This effect would be mediated by increasing pyramidal neuron activity through blockade of 5-HT3 receptor-mediated activation of GABA interneurons. Our own research group demonstrated that the coadministration of a 5-HT3 antagonist with selective serotonin reuptake inhibitors (SSRIs) increased monoamine levels in the rat frontal cortex. Moreover, it was suggested that the blockade of 5-HT3 receptors in coadministration with SSRIs could be a potential strategy for avoiding counter-therapeutic effects of SSRIs in the early stages of treatment.

Mirtazapine and the new antidepressant vortioxetine are examples of antagonists of the 5-HT3 receptors. Vortioxetine presents 5-HT3 receptor antagonism and selective serotonin reuptake inhibition in the same molecule. Several studies with vortioxetine have demonstrated that the 5-HT3 receptor antagonism potentiated the increase in 5-HT extracellular levels produced by the serotonin reuptake inhibition.

**Key words:** 5-HT3 receptors, depression, antidepressants, vortioxetine

## S9-3
PHARMACOLOGY OF NEW PSYCHOACTIVE SUBSTANCES

Farré M.¹, Torrens M.², Papaseit E.¹

¹Hospital Universitari Germans Trias i Pujol (HUGTP-IGTP) and Universitat Autònoma de Barcelona, Barcelona, Spain; ²Institut de Neuropsiquiatria i Addicions, Parc de Salud Mar and Universitat Autònoma de Barcelona, Barcelona, Spain

The use of new psychoactive substances (NPS) has increased in recent years. Until December 2016, more than 700 NPS had been reported by 102 countries to United Nations since 2008. NPS are defined as "substances of abuse, either in a pure form or a preparation, that are not controlled by the 1961 Single Convention on Narcotic Drugs or the 1971 Convention on Psychotropic Substances, but which may pose a public health threat". The term "new" does not necessarily refer to new inventions, but to substances that have recently become available on the market. Since NPS are not controlled their legal status can differ widely from country to country. NPS are cheap, easy to obtain, and often legally available. NPS appeared in the market by terms such as "legal highs", "herbal highs", "bath salts" and "research chemicals". The main substance groups of NPS are synthetic cannabinoids, phenethylamines (including 2C-derivatives), synthetic cathinones (including mephedrone), aminoindanes, ketamine and phencyclidine-type substances, piperazines, tryptamines, benzodiazepines, opioids and plant-based substances.

In general, side effects of NPS range from seizures to agitation, aggression, acute psychosis and cardiovascular effects. NPS users have frequently been hospitalized with severe poisonings including cases of death. Information about pharmacology, safety and toxicity in animals and humans of most NPS are not available or very limited, in addition, long-term adverse effects or risks are still largely unknown.

In this conference, recent results of the presence of NPS in the Spanish market and the human pharmacology of mephedrone, 2C-B and synthetic cannabinoid JWH-018 will be presented and discussed.

**Acknowledgements:** Founded in part by grants from Instituto de Salud Carlos III (ISCIII, FIS-FEDER, PI14/00715), ISCIII-Red de Trastornos Adictivos (RTA RD16/0017/0003 and RD16/0017/0010), Ministerio de Sanidad, Servicios Sociales e Igualdad (Plan Nacional sobre Drogas-PNSD, 2015I054). Esther Papaseit is a Juan Rodes fellow (ISC-III, JR16/00020).

**Key words:** New psychoactive substances, synthetic cathinones, synthetic cannabinoids, phenethylamines, intoxication
# ROLE OF INFLAMMATORY PROCESSES IN DEPRESSION

*Capuron L.*

Laboratory of Nutrition and Integrative Neurobiology, INRA –
University of Bordeaux, Talence, France

A large dataset documents the role of immune-to-brain interactions in the pathophysiology of depressive disorders. Inflammatory cytokines released peripherally by activated immune cells during the host response to pathogen invasion play a major role in these interactions. To further elucidate the role of inflammatory processes in the

development of depression, our group has used models of chronic inflammation, both at the clinical and preclinical levels. Findings have contributed to characterize the phenomenology of depressive symptoms that occur in contexts of inflammation and to describe the underlying mechanisms and biomarkers associated with these effects. In particular, they revealed the key contribution of alterations in enzymatic pathways involved in the biosynthesis of neurotransmitters, together with changes in neuroendocrine activity and neurocircuitry. Altogether, these data provide important information regarding the role of immune mediators in the pathophysiology of depression and highlight potential new therapeutic targets.
# S10: THERAPEUTIC INNOVATION

## S10-1
P2Y12 INHIBITION IN ACS: PLATELET AND BEYOND PLATELET EFFECTS

Badimon L.¹,², Mendieta G.², Casani L.¹,², Vilahur G.¹,²

¹Cardiovascular Sciences Institute, IIB-SantPau, Hospital de la Santa Creu I Sant Pau, CiberCV, Spain; ²Department of Cardiology, Hospital Clinic, Barcelona, Spain

P2Y₁₂ receptor antagonists have potent effects inhibiting the ADP-induced activation of platelets. Dual antiplatelet therapy with the cyclo-oxygenase (COX) inhibitor aspirin and a purinergic P2Y₁₂ receptor inhibitor is at present the standard of treatment in acute coronary syndromes (ACS). Different clinical trials have shown the relative efficacy of clopidogrel, prasugrel and ticagrelor in ACS patients. Recently it has become increasingly accepted that P2Y₁₂ inhibitors exert off-target effects that protect against myocardial injury. Yet, the extent of cardioprotection differs among the different P2Y₁₂ antagonists likely because their off-target properties. As such, administration of P2Y₁₂ inhibitors has demonstrated, at both experimental and clinical levels, to reduce the size of infarction. This is of key importance since infarct size is a major determinant of post-STEMI morbidity and mortality. However, P2Y₁₂ inhibitors have been shown to differ in their ability to limit the size of infarction regardless of their antiplatelet efficacy. In this regard, we have recently demonstrated in a preclinical animal model of MI-induction and by CMR analysis, that although clopidogrel and ticagrelor administration prior-MI reduces the size of infarction assessed acutely post-MI, this effect was significantly greater in ticagrelor-administered animals. Most interestingly, ticagrelor cardioprotective effects were associated with the attenuation in oedema formation, an effect not observed in clopidogrel-treated pigs. These differences suggest that P2Y₁₂-related cardioprotection may exhibit a class effect (clopidogrel/thienopyridine vs ticagrelor/cyclopentyl triazolopyrimidine) helping to partly explain the differences in the clinical benefit found in the PLATO trial. Although the underlying molecular mechanisms by which platelet P2Y₁₂ inhibitors mediate cardioprotection remain to be elucidated they have been shown to exhibit an ischemic conditioning-like effect. Particularly, ticagrelor has shown to protect the heart against MI though adenosine-related mechanisms. By blocking the ENT-1 transporter, ticagrelor prevents erythrocytes re-uptake of the adenosine released upon the ischemic insult leading to the consequent local adenosine increase. In a follow-up study we have demonstrated that ticagrelor protects also against adverse cardiac remodelling.

**Key words:** platelets, ACS, cardioprotection, ticagrelor.

## S10-2
PHARMACOECONOMICS APPLIED TO BIOLOGICAL DRUGS IN THE MANAGEMENT OF SEVERE ASTHMA

Martinez Moragon E.

Department of Pneumology, University Hospital Dr Peset, Valencia, Spain

Asthma currently affects around 300 million people globally, being one of the most prevalent chronic diseases, affecting 5% of the adult population in Spain. Approximately 10% of patients with asthma suffer from severe asthma, which has an increased risk of death. Achieving good disease control involves taking good control of symptoms and reducing the number of exacerbations. Approximately 50% of patients with severe asthma receiving maintenance therapy, through the combination of inhaled corticosteroids (IC) and long-acting β₂-adrenergic agonists (LABA) do not achieve good disease control, as evaluated through the administration of the Asthma Control Questionnaire. In addition, these poorly controlled patients are the ones that generate the greatest cost to the National Health System and those that show a greater affectation of the quality of life. The management of these patients in specialized units of asthma is cost-effective (improving asthma control and reducing exacerbations and hospitalizations) and this is achieved with therapeutic modifications that sometimes include addiction to the treatment of biological drugs. Biological therapy in severe asthma has a beneficial impact on disease control, provided that patients are adequately selected. The results obtained in the published studies suggest that severe asthmatics treated with biological agents (after appropriately selecting the appropriate therapeutic target for each patient) can improve their symptoms and increase the number of QALYs, consume less health resources and have a lower loss of productivity. All these variables should be considered in real practice when choosing an effective treatment and improving the quality of life of patients with severe asthma.

**Key words:** uncontrolled severe asthma; biological drugs; quality of life; pharmacoeconomics.

## S10-3
TARGETED IMMUNOTHERAPY FOR CANCER TREATMENT, DISCOVERING A NEW ERA IN ONCOLOGY

De la Cruz Merino L.

Jefe de Servicio de Oncología Médica, Hospital Universitario Virgen Macarena, Seville, Spain

In the last few years modern immunotherapy has emerged as a powerful tool to successfully treat a large number of solid and hematologic malignancies. Monoclonal antibodies targeting immunomodulatory proteins of the immune synapses like cytotoxic T-lymphocyte associated protein 4 (CTLA-4) or programmed cell death protein 1 (PD-1) represent a breakthrough therapy that have reported impressive results in a wide set of different tumours such as melanoma, lung, kidney, head and neck cancer, Hodgkin lymphoma and others. Nowadays, this topic is an area of expanding clinical and translational research, with uncountable clinical trials ongoing. However, despite the awesome results achieved, a considerable fraction of patients does not respond to immunotherapy and thus the great challenge that lies ahead is to identify predictive biomarkers that would allow using these new therapies in the patients most likely to benefit, and therefore using them in a more efficient way.
# S11: GASTROINTESTINAL AND HEPATIC PHARMACOLOGY

## S11-1
THE IMPORTANCE OF THE TRANSPORTOME IN HEPATIC AND GASTROINTESTINAL DISEASES

*Pastor-Anglada M.*

Institute of Biomedicine of the University of Barcelona (IBUB) and CIBER EHD, Barcelona, Spain

Epithelial cells express a broad variety of membrane transporter proteins which coordinately determine nutrient supply but also modulate other tissue functions, including purinergic responses and cell proliferation. These membrane transporter proteins, understood as a whole functionally integrated system is known as the transportome. Transportome alterations may be relevant to the physiopathology of particular diseases but can also determine response to therapy. Transportome alterations in Crohn's disease with a particular emphasis on the purinome will be discussed. Purinergic signaling in inflammation is in part modulated by selected membrane transporters implicated in adenosine removal. How the microbiota can contribute to restore transportome integrity will be also briefly summarized. Similarly, the network of transportome encoding genes in gastrointestinal tumor cell models will be also reviewed, focusing on how selected genes in these networks may be modulating response to selected anticancer drugs. Although alterations in transporter membrane proteins can indeed determine drug bioavailability and action, recent evidence supporting the view that loss of selected transportome members may contribute to oncogenesis will be also briefly reviewed.

## S11-2
PHARMACOLOGICAL TARGETS FOR NASH

*Martínez C., Luz M.*

University of the Basque Country, CIC bioGUNE, CIBERehd – Spanish Carlos III Health Institute, Vizcaya, Spain

NAFLD is one of the most common causes of chronic liver disease worldwide, and particularly, the main chronic liver disease condition in the Western world. NAFLD is a clinical pathological term that includes a spectrum of alterations that range from simple triglyceride accumulation in the hepatocytes (steatosis) to hepatic steatosis with inflammation (nonalcoholic steatohepatitis or NASH), which may or not have associated fibrosis. NAFLD is tightly linked to obesity, insulin resistance, type 2 diabetes, dyslipidemia and hypertension, all of them risk factors to the metabolic syndrome. Metabolic syndrome is a clustering of different risk factors that collectively increases the probability of developing cardiovascular disease and diabetes. NAFLD can be considered the hepatic manifestation of the metabolic syndrome, being a key factor predisposing to it.

The prevalence of NAFLD in developed countries is 20-30% of adults. It is more frequent among people with diabetes (30-50%) and obesity (80-90%), and almost universal when combining both factors. In the case of children, there is a prevalence of 3-10% rising up to 40-70% among obese children. Even though NAFLD is the most common liver disorder in developed countries, current therapies are restricted to lifestyle modifications whereas pharmacological approaches remain scarce and experimental. Indeed, it has been shown that 7-10% of weight loss slows the progression of NAFLD and may reverse some of the steatosis and necroinflammation. There are few clinical trials in NAFLD and no specific therapy can be firmly

recommended. For this reason, drug therapy should be reserved for patients with progressive NASH or with high risk of disease progression (diabetes, metabolic syndrome, persistently increased ALT or high necroinflammation) to prevent disease progression.

Our group have shown that knocking out enzymes involved in S-adenosylmethionine (SAMe) metabolism, the main biological methyl donor in humans that is abundant in the liver, will lead to NASH development in mice. New data from humans have also suggested that these enzymes play a role in the pathogenesis of NAFLD and that some of SAMe cycle metabolites may serve as noninvasive biomarkers of NASH and potentially as NASH treatment. Novel druggable therapeutic targets will definitely impact the huge health and economic burden NAFLD-related.

## S11-3
IMMUNE THERAPY OF LIVER CANCER

*Sangro B.*

Liver Unit, Clínica Universidad de Navarra-IDISNA-CIBEREHD, Pamplona, Spain

Several approaches to enhance the immune response against tumor cells have failed to provide treatment benefit in advanced hepatocellular carcinoma (HCC) including dendritic cell vaccination, cytokine-induced killer cells or oncolytic viruses expressing immunostimulating cytokines. Following the proof of concept in melanoma patients, it has been shown that T-cell checkpoint modulation may break the barrier that different tumor types create to evade the attack from the immune system.

In fact, enhancing T cell function by targeting cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) using monoclonal antibodies has revolutionized the field of cancer immunotherapy. Tremelimumab (anti-CTLA-4) has provided the first signal of activity and favorable safety profile in a phase II clinical trial targeting HCC patients with chronic HCV infection. In a small group of 21 patients, Tremelimumab produced an overall response rate of 18% while 59% of patients had a stable disease that in almost half of the cases lasted for more than 6 months. A median time to progression of 6.5 months provided further evidence of antitumor activity. These results paved the way for other checkpoint inhibitors.

Nivolumab (anti-PD-1) has very recently provided strong signal of antitumor activity in a phase I/II clinical trial. A dose-escalation cohort was followed by a large expansion cohort, and uninfected patients as well as patients with chronic HCV infection or chronic HBV infection under control by antiviral agents were recruited worldwide. Altogether, a large population of nearly 250 patients with fairly advanced HCC has been treated, most of them already exposed to Sorafenib. Following Nivolumab, 15% of patients reached an objective tumor remission (including several complete responses) and an additional 50% had stable disease that was frequently durable. Regarding survival, a 9-month survival rate of 70% was reported in the large expansion cohort while a median overall survival of 14 months (irrespective of prior Sorafenib treatment) was reported in the dose-escalation cohort with a longer follow-up. Importantly, these results were obtained with only a 1% rate of intense (CTCAE grade ≥3) symptomatic adverse events. Other checkpoint inhibitors have started clinical development and, following reports of synergistic effects in melanoma, a lot of interest is focused on combinations. CTLA-4 plus PD-1 or PDL-1 blockade is
being explored in two different trials using the combination of Ipilimumab plus Nivolumab or Tremelimumab plus Durvalumab, and their results are eagerly awaited.

S11-4
PURINE RECEPTORS AND SMOOTH MUSCLE FUNCTION IN THE GASTROINTESTINAL TRACT

Jimenez M.

Universitat Autònoma de Barcelona, Institut de Neurociències and CIBERehd, Barcelona, Spain

Gastrointestinal motility (GI) is accomplished by the interaction between the enteric nervous system and pacemaker cells. Enteric neurons, glial cells and interstitial cells of Cajal (ICC) interact with smooth muscle to coordinate motor patterns such as gastric emptying, propulsion, segmentation and sphincter accommodation among others. Purine receptors including several subclasses of P2X and P2Y receptors are present in different cell types and modulate gastrointestinal motility. They are both pre- and post-junctional located and can contribute to the modulation of nerve mediated excitatory and inhibitory

responses. Nerve-mediated relaxation is accomplished by Nitric Oxide (NO) and ATP or a related purine that are probably released by the same inhibitory motor neuron as inhibitory co-transmitters. The P2Y₁ receptor has been recently identified as the receptor responsible for purinergic smooth muscle hyperpolarization and relaxation in the gut. Both orthosteric (MR2179, MRS2279 and MRS2500) and allosteric (BPTU) P2Y₁ antagonists concentration dependently decrease nerve-mediated inhibitory responses. Purinergic neurotransmission is absent and transit time is impaired In P2Y₁-deficient mice. The dynamics of the co-transmission process is frequency dependent: low frequencies of nerve stimulation cause P2Y₁ mediated responses whereas high frequencies of stimulation cause NO responses, suggesting a different role for each co-transmitter. A direct communication from nerve to muscle is possible. However, specific cell types such as ICC or fibroblast-like cells expressing PDGFRα and P2Y₁ receptors are intercalated between nerve and muscle. Accordingly inhibitory neurons modulate smooth muscle contractility through other cells that are coupled each other and contribute to pacemaker function. The contribution of purinergic neurotransmission in neuromuscular diseases affecting GI motility is still not understood. In this presentation, we will focus on the physiological mechanisms responsible for nerve-mediated purinergic relaxation providing the functional basis for possible future clinical and pharmacological studies on GI motility targeting purine receptors.
# S12: TEACHING PHARMACOLOGY

## S12-1
ARE TV MEDICAL DRAMAS USEFUL TO TEACH CLINICAL PHARMACOLOGY? AN EMPIRICAL STUDY USING HOUSE M.D.

Baños J.E.¹, Farré M.², Lucena M.I.³

¹GRECS, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain; ²Department of Pharmacology, Therapeutics and Toxicology, Universitat Autònoma de Barcelona, Hospital Universitari Germans Trias i Pujol, Badalona, Spain; ³Clinical Pharmacology Service, IBIMA, HU Virgen de la Victoria, University of Málaga, Málaga, Spain

TV series with medical plots have always had a great success in general public and they have been used as a teaching tool in medical students. However, their use has not reached clinical pharmacology courses and empirical data do not exist about their pedagogical effectiveness. Therefore, we performed an initial content analysis study to evaluate the 22 chapters of the first season of *House MD*. Later, we carried out a multicenter study in three universities to assess its pedagogical value to learn clinical pharmacology in medical students. To this aim, we compared how seeing and discussing a chapter improved the factual knowledge. Students were randomly assigned to two groups, each of them seeing a different episode, and answered a multiple choice questionnaire that included questions related to both chapters. Finally, we asked the students to rate the interest of the experience for learning the discipline. The content analysis showed that 43 primary and 136 secondary issues likely to be of interest in teaching clinical pharmacology were chosen. The evaluation of the pedagogical interest identified five episodes of great interest, 13 of medium interest and four low interest. Four episodes of the first group were used in the comparative study. The evaluation with students showed that two chapters allowed the improvement of knowledge in all universities, one was not useful in any of them and contradictory results were seen with the fourth. However, students showed a high satisfaction with their experience and the interest in improving their knowledge in all study groups. In conclusion, the project showed that *House MD* contains elements of pedagogical interest in the field of clinical pharmacology. The view of some episodes allowed to obtaining a better knowledge of the discipline but not in all cases. Consequently, its use should consider prior knowledge when choosing the episode.

## S12-2
SPANISH INTER-UNIVERSITY NETWORK FOR INNOVATIVE EDUCATION IN PHARMACOLOGY: A COMMON SPACE TO IMPROVE LEARNING?

Ferrándiz M.L., D'Ocón P., Ivorra M.D.

Pharmacology Department, University of Valencia, Valencia, Spain

Changes in Pharmacology teaching have been introduced in the last years that include not only changes in the curriculum but also in the way teaching is delivered to facilitate learning. In this context, building materials for innovative education in Pharmacology is a task that needs to be envisaged. Until now, problem based learning, simulated practical works, peer assessment, interactive computer learning, virtual environments and multidisciplinary activities have been implemented in different universities, but collaboration between teachers on a global scale could be an effective strategy to optimize the necessary changes to improve learning.

We initiated a Spanish inter-university network to create a common space for sharing not only the teaching material but mainly the implementation experiences and research results obtained on different aspects of innovation. Fourteen Spanish universities that teach pharmacology to different Degrees of Health Sciences are associated to this project. A Moodle platform has been organized, presented to the network members in a meeting and used to share teaching materials. Until now, shared material includes: pharmacological cases and problems for seminars or practical sessions, videos for flipped class or as an alternative of animal use, discussion activities based on films or medical series visualization or literary texts and press news reading, use of social network, gamification, ...as well as other resources to practical sessions as computer-based simulation software, practice notebooks. Another shared resources were the different evaluation and self-assessment systems used in each institution. Along with the teaching material, a description with the experience of its use and the evaluation of results, have been added in the Moodle interface.

With the experience accumulated during the last and the present year, we will evaluate the contribution of the network to the improvement of results in pharmacological education.

Funded by Vice-Rectorate for Training Policies and Educational Quality (UVEG) (UV-SFPIE_GER16-418743).

**Key words:** Interuniversity network; pharmacology teaching; innovation

## S12-3
AN EXPERIENCE OF USING E-LEARNING IN THE TEACHING OF PHARMACOLOGY

Sánchez S., Carbonell L., Ciruela F., Cuffí L., Fernández-Dueñas V., Fernández A., Vallano A.

Pathology and Experimental Therapeutics, Universitat de Barcelona, Barcelona, Spain

The aim of our experience consists of developing E-learning strategies to carry out a functional teaching in Pharmacology. The student should be able to use the acquired knowledge in their profession. Instead of retaining information that would remain inert or forgotten, they must perform activities to allow them using the knowledge of Pharmacology to solve real situations. A new E-learning tool based on thematic units was implemented; students achieve theoretical knowledge and develop skills such as collaborative work and self-learning, which increases if thematic units are delivered before theoretical class.

The application of the thematic units, before the theoretical class increases the degree of self-study with a similar academic performance, when thematic units is working after class attendance.

The assessment of the methodology by the students is very positive, mostly expressing their utility when not only in the resolution of enforcement activities (situations similar to real clinical cases), but also with regard to the possibility of working group.

We have prepared a video that shows the methodology used to conduct E-learning activities and assessment activities, wishing that can be useful to other teachers.

The video is in the following sections: introduction, presentation of the methodology for students and formation of working groups, theoretical class, working group session, interviewed customized for students to obtain its opinion on the methodology and conclusions.

*This work was carried out with the aid granted by the UB our Consolidated Teaching Innovation Group (GINDOC-UB/094)

**Key words:** e-learning; functional teaching; self-learning; collaborative work
S12-4
AN ACADEMIC SERVICE-LEARNING ACTIVITY IN HIGHER
EDUCATION TO PROMOTE THE RESPONSIBLE USE OF
ANTIBIOTICS

Fernández A.¹, Ignatova T.¹, Yashchenko O.¹, González M.²,
Granados V.², Morgó J.², Sabaté M.², Salvat B.², Solé J.², Trullàs E.²,
Vacas N.², Vallés A.¹, Vilaseca J.-E.², García M.À.², Cuffí M.L.¹

¹Faculty of Medicine and Health Sciences, Barcelona, UK; ²Faculty of Library and Information Science, Universitat de Barcelona, Barcelona, Spain

Service-learning is an educational approach that integrates students' academic learning with community service. The aim of this service-learning activity is to promote a responsible use of antibiotics and to improve students' academic learning related to their own contents of Pharmacology and Audiovisual Projects as well as the ones related to transversal competences. The service-learning activity began with the identification of a real need: to avoid the spread of antimicrobial resistance due to the irresponsible use of antibiotics. Next, Medical students had to prepare their plan for action: to encourage best practices amongst the general public and health workers about antibiotics

consumption. An audiovisual media campaign was chosen in order to
improve awareness and understanding of antimicrobial resistance. For
that purpose, it was necessary to establish a partnership with the stu-
dents of the Degree in Audiovisual Studies. With this action, it was
expected students to learn how to communicate scientific subjects in a
language style appropriate to the subject and audience, to detect some
common mistakes in antibiotics prescription or therapeutic compliance,
to make audiovisual and multimedia productions and to acquire some
abilities to network with experts from another field of knowledge. Stu-
dents analysed the most suitable information about antibiotic consump-
tion to be included in the audiovisual product and chose the
audiovisual product that best fit the sector of the population targeted.
Moreover, an agreement with a cooperation entity was signed. After
the action was made, a period of reflection started. The experience,
knowledge and skills acquired were evaluated for the students. How-
ever, reflection could also occur before, during and after implementa-
tion of the action. Finally, service-learning project was globally
assessed: the extent to which the initial objectives have been accom-
plished.

Kaye, C. B. *The complete guide to service learning: Proven, practical ways to engage students in civic responsibility, academic curriculum, & social action.* Free Spirit Publishing, 2004.
# S13: NEUROPHARMACOLOGY

## S13-1
VULNERABILITY FACTORS THAT PREDISPOSE TO COCAINE ADDICTION

García-Fuster M.J.

IUNICS, University of the Balearic Islands, and IdISBa, Palma de Mallorca, Spain

A large number of people experiment with drugs of abuse, but only a few become addicts. This individual variation in the propensity to become addicted is driven by both genetic and environmental factors. Many studies have investigated vulnerability factors that predispose to cocaine addiction, but few have focused on adolescence—a critical period for neurobehavioral plasticity and vulnerability to drug use. This symposium will present pre-clinical data from our laboratory which demonstrated timing-specific cocaine exposure effects during early-mid adolescence (i.e., vulnerable window: postnatal day PND 33–39) on inducing immediate neurotoxic effects on particular brain regions and long-term consequences on addiction-like behavior (i.e., stimulus-reward learning) and negative affect (i.e., psychiatric comorbidity). Supported by ‘Delegación del Gobierno para el Plan Nacional sobre Drogas’ (MSSSI, Grants 2012/011, 2016/002) and ‘Ramón y Cajal’ Program to M.J.G.-F. (MINECO), and by RTA-RD16/0017/0010.

**Key words:** adolescence; cocaine; long-term consequences; negative affect

## S13-2
POTENTIAL VALUE OF MICRORNA-30C AS BIOMARKER AND THERAPEUTIC TARGET FOR NEUROPATHIC PAIN

Tramullas M.¹,², Francés R.¹,², de la Fuente R.²,³, Velategui S.¹, Hurlé M.A.¹,²

¹Departamento de Fisiología y Farmacología, Facultad de Medicina, Universidad de Cantabria, Santander, Spain; ²Instituto de Investigación Valdecilla (IDIVAL), Santander, Spain; ³Servicio de Anestesiología y Reanimación, Hospital Universitario Marqués de Valdecilla, Universidad de Cantabria, Santander, Spain

Neuropathic pain is a prevalent and debilitating chronic syndrome highly refractory to current analgesics. MicroRNAs (miRNAs) are single-stranded ~22nt long non-coding RNA transcripts involved in the post-transcriptional regulation of gene-expression. Recently, a number of studies have reported aberrant expressions of several miRNAs in nociception-related structures, which are associated to neuropathic pain development in rodent models. Herein, we have exploited the anti-allo-dynic phenotype of mice lacking BAMBI (BMP and Activin membrane bound inhibitor) to identify, by next-generation sequencing, new miRNAs potentially valuable as therapeutic targets and/or biomarkers for neuropathic pain states. Among them, miR-30c overexpression in pain-relevant areas (spinal cord, dorsal root ganglion) and body fluids (cerebrospinal fluid (CSF) and plasma) showed a direct correlation with the severity of the mechanical allodynia developed by rats subjected to sciatic nerve injury. Administration of LNA miR-30c inhibitor into the cisterna magna delayed neuropathic pain development and reversed fully established allodynia. In contrast, administration of miR-30c mimic accelerated allodynia development after the nerve injury. After witnessing the relevant contribution of miR-30c to neuropathic pain in rats, we aimed to assess the potential translation of the experimental data into humans. For this purpose, miR-30c was determined in plasma and CSF samples from patients suffering from neuropathic pain associated to severe leg ischemia. Patients with no evidence of

neuropathic pain, with either leg ischemia or without ischemia served as controls. As observed in the experimental animal, patients affected by neuropathic pain presented significantly higher levels of miR-30c in both plasma and CSF in comparison with patients free of pain. Logistic regression analysis and receiver operating characteristic (ROC) curves identified circulating miR-30c, either in plasma or in CSF, diabetes mellitus and age as significant positive predictors of neuropathic pain occurrence. The predictive model obtained if diabetes and age were combined with circulating miR-30c in plasma presented the highest area under the ROC curve [AUC = 0.93 (95% CI 0.87–0.99)], with a sensitivity of 80% and a specificity of 94%. Overall, our results support that miR-30c is involved in neuropathic pain establishment and maintenance after neural injuries. Our findings in patients support the notion that the level of circulating miR-30c, either in plasma or in CSF, in conjunction with clinical parameters, could help to create an individual risk profile disclosing the potential for neuropathic pain in patients suffering from chronic peripheral ischemia. We propose that (i) targeting miR-30c could constitute a therapeutic approach in chronic pain processes and (ii) miR-30c circulating levels could be useful biomarkers of disease prognosis. (Supported by SAF2013-47434-R).

## S13-3
BEHAVIORAL AND MOLECULAR ALTERATIONS INDUCED BY MATERNAL ALCOHOL BINGE CONSUMPTION IN OFFSPRING MICE

Valverde O., Cantacorps L.

Neurobiology of Behavior Research Group (GReNeC), Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain

Maternal alcohol consumption leads to a range of long-lasting morphological and behavioral deficiencies known as fetal alcohol spectrum disorders, associated with neurodevelopmental disabilities. Alcohol activates neuroimmune system that is a critical contributor to brain injury outcome. We sought to test whether alcohol binge drinking during the prenatal period, or in combination with lactation, promotes long-term behavioral alterations and whether such effects are associated with neuroinflammation and myelin dysfunctions. Pregnant C57BL/6J female mice underwent a model procedure for binge alcohol drinking either during the gestation period or both the gestation and lactation periods. Subsequently, adult male offspring were assessed for their cognitive function and motor coordination. Recognition memory was not significantly affected by maternal binge alcohol drinking, but executive functioning was impaired in prenatal and lactation alcohol-exposed mice. The behavioral effects were associated with up-regulation of pro-inflammatory molecules (Toll-like receptor 4, Nuclear factor-kappa B p65 and Interleukin-1β) together with a reduction in several structural myelin proteins (MAG, MBP, PLP) in both the prefrontal cortex and hippocampus of adult mice exposed to alcohol. Finally, histone acetylation levels (H3K9, H4K5 and H4K12) and an imbalance in histone acetyltransferase and histone deacetylase activities were found to increase in the prefrontal cortex of both groups of mice exposed to alcohol, with more discrete effects in the hippocampus. Altogether, our results reveal that maternal binge-like alcohol consumption induces neuroinflammatory damage causing long-term myelin and epigenetic modifications, effects that might underlie the long-term cognitive and behavioral impairments observed in fetal alcohol spectrum disorders.

**Key words:** fetal alcohol spectrum disorders, neuroinflammation, myelin, mice
S13-4
THERAPEUTIC POTENTIAL OF CONJUGATED SIRNAS FOR
THE TREATMENT OF MAJOR DEPRESSIVE DISORDER

*Artigas F.*¹,²,³, *Bortolozzi A*.¹,²,³

¹Department of Neurochemistry and Neuropharmacology, IIBB-CSIC (Consejo Superior de Investigaciones Científicas), Barcelona, Spain;

²Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; ³Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), ISCIII, Madrid, Spain

The high prevalence and socioeconomic impact of major depressive disorder (MDD) is not paralleled by marked improvements in its treatment. Antidepressant drugs, including the widely prescribed monoamine (serotonin-5-HT and norepinephrine-NE) reuptake inhibitors, show slow onset of clinical action and limited efficacy, which reduces the quality of life of MPP patients and increases suicide risk. The reasons for the limited efficacy of monoamine-based drugs are manifold, including neuroadaptive mechanisms and genetic factors.

RNAi strategies may avoid some of these limitations by directly modu-
lating the expression of genes involved in clinical response. However,
despite its enormous potential, in vivo use of RNAi is limited due to
the difficulty to deliver oligonucleotide sequences (e.g., small interfer-
ing RNA; siRNA) to the desired neurons/circuits in mammalian brain.
Our strategy has been to develop conjugate siRNA molecules (C-
siRNA) in which the siRNA sequence is covalently bound to the SSRI
sertraline in order to selectively accumulate it by the dense network of
5-HT axon terminals in brain. Hence, the intracerebroventricular
(i.c.v.) or intranasal (i.n.) administration of the sertraline-conjugated
siRNA (Ser-C-siRNA) directed against the serotonin transporter
(SERT) or 5-HT1A-autoreceptors evoked robust antidepressant-like
responses in mice,¹,² mimicking the effects of the local application of

unmodified siRNA in the raphe nuclei.³,⁴ Interestingly, 1-week i.n. administration of small amounts (2.1 nmol/day) of a Ser-C-SERT-siRNA evoked antidepressant-like responses similar to those induced by 1-month treatment with 10 mg/kg/day fluoxetine. Likewise, acute 5-HT₁A autoreceptor knockdown also evoked rapid antidepressant-like effects in mice, due to a lesser self-inhibition of serotonergic neurons. We recently extended this strategy to knockdown TASK3, an acid-sensitive two pore-domain potassium channel (K2P) selectively in serotonergic or noradrenergic neurons using sertraline- and reboxetine-conjugated siRNA, respectively.

Overall, these results support the usefulness of our approach to elicit
rapid and robust antidepressant-like effects in rodents using RNAi,
showing a high potential translational value.

References:

1. Bortolozzi A, Castañé A, Semakova J, Santana N, Alvarado G, Cortés R et al (2012). Selective siRNA-mediated suppression of 5-HT<sub>1A</sub> autoreceptors evokes strong anti-depressant-like effects. *Mol Psychiatry* 17: 612–623.

2. Ferrés-Coy A, Galofré M, Pilar-Cuéllar F, Vidal R, Paz V, Ruiz-Bronchal E et al (2016). Therapeutic antidepressant potential of a conjugated siRNA silencing the serotonin transporter after intranasal administration. *Mol Psychiatry* 21:328–338.

3. Ferrés-Coy A, Santana N, Castañé A, Cortés R, Carmona MC, Toth M et al (2013). Acute 5-HT1A autoreceptor knockdown increases antidepressant responses and serotonin release in stressful conditions. *Psychopharmacology* (Berl) 225: 61–74.

4. Ferrés-Coy A, Pilar-Cuellar F, Vidal R, Paz V, Masana M, Cortés R, Carmona MC, Campa L, Pazos A, Montefeltro A, Valdizán EM, Artigas F, Bortolozzi A (2013) RNAi-mediated serotonin transporter suppression rapidly increases serotonergic neurotransmission and hippocampal neurogenesis. *Transl Psychiatry* 3:e211
# S14: PHARMACOLOGY OF ANTI-CANCER DRUGS

## S14-1

### ABTL0812 AN AUTOPHAGY-ANTICANCER DRUG IN PHASE II CLINICAL TRIALS THAT INHIBITS PI3K/AKT/MTOR PATHWAY BY INDUCING TRIB3 EXPRESSION

Alfón J.¹, Erazo T.², Pérez-Montoyo H.¹, Domènech C.¹, Puig T.³, Segura M.F.⁴, Matías-Guiu X.⁵, Rodon J.⁶, Nadal E.⁷, Oaknin A.⁶, Gil M.⁷, Vidal L.⁸, Gascon P.⁸, Lizcano J.M.²

¹Ability Pharmaceuticals SL, Barcelona, Spain; ²Universitat Autònoma de Barcelona, Barcelona, Spain; ³Universitat de Girona, Girona, Spain; ⁴Vall d'Hebron Institute of Research, Barcelona, Spain; ⁵Hospital Universitari de Bellvitge and Hospital Arnau de Vilanova de Lleida, Lleida, Spain; ⁶Vall d'Hebron Institute of Oncology, Catalonia, Spain; ⁷Institut Català d'Oncologia, Barcelona, Spain; ⁸Hospital Clínic de Barcelona, Barcelona, Spain

ABTL0812 is a first-in-class anti-cancer agent with a unique mechanism of action via a PPARα/γ-dependent TRIB3-mediated AKT/mTOR pathway inhibition that leads to autophagy-induced cell death. The antitumour activity of ABTL0812 has been demonstrated in *in vitro* models including squamous and adenocarcinoma non-small cell lung cancer, endometrial cancer, pancreatic cancer, breast cancer and prostate cancer, even in treatment-resistant cellular models such as doxorubicin-breast cancer resistant cells. *In vivo* studies showed that ABTL0812 reduced tumour growth to a similar or greater extent than erlotinib in NSCLC cells. Notably, when ABTL0812 was administered in combination with standard-of-care chemotherapies, it exhibited increased antitumour activity in different models such as docetaxel or paclitaxel/carboplatin in squamous NSCLC xenograft models, paclitaxel in an orthotopic endometrial cancer model, paclitaxel/carboplatin in an endometrial PDX model, paclitaxel/gemcitabine in a pancreatic cancer xenograft model or cisplatin in a neuroblastoma xenograft model. In these cases, the efficacy of the combined therapy was higher than that observed for the single agents; however, no potentiation of toxic effects was observed.

All this preclinical work led to the first-in-human dose escalation phase I clinical trial where ABTL0812 was administered as a single agent in adult patients with advanced solid tumours. The study included twenty-nine patients and showed that ABTL0812 has an excellent safety profile. AKT phosphorylation in platelets was used as a surrogate pharmacodynamic biomarker that permitted the determination of the recommended phase 2 dose by pharmacokinetic-pharmacodynamic modelling. Additionally, preliminary signs of efficacy were observed in five patients presenting with stable disease, including one cholangiocarcinoma (80 weeks), one endometrial cancer (60 weeks) and three sigmoid colon adenocarcinomas (28 and 22 and 20 weeks). A phase 2 clinical trial is currently ongoing in patients with endometrial or squamous NSCLC cancer where ABTL0812 is administered as first-line therapy in combination with standard-of-care chemotherapy.

**Key words:** ABTL0812, autophagy, PI3K/Akt/mTOR pathway, Phase I/II, lung cancer, endometrial cancer

## S14-2

### NEW THERAPEUTIC PROPOSALS EMERGING FROM PATIENT-DERIVED PRECLINICAL PLATFORMS OF PEDIATRIC SOLID TUMORS

Carcaboso A.M.

Institut de Recerca Sant Joan de Déu, Barcelona, Spain

Most pediatric solid tumors are characterized by homogeneous genetic properties and unique biomarkers. Examples of specific alterations of pediatric tumors are the high expression of the disialoganglioside (GD2) in neuroblastoma and osteosarcoma, the EWS/FLI1 fusion gene of Ewing sarcoma, or the K27M mutation in the H3.3 histone of diffuse intrinsic pontine gliomas. These unique properties enable the design of novel strategies to target pediatric tumor cells. However, preclinical studies in pediatric oncology are challenging because pediatric solid tumor models are scarce. The translational research program at Hospital Sant Joan de Déu Barcelona contributes to generate clinically relevant models of pediatric solid tumors, including patient-derived xenografts, which are useful for the validation of novel therapeutic approaches, the identification of biomarkers and the design of clinical trials.

## S14-3

### THE CANCER GENOME INTERPRETER: A TOOL TO ANNOTATE THE BIOLOGICAL AND CLINICAL RELEVANCE OF TUMOR ALTERATIONS

Tamborero D.¹,²

¹Biomedical Genomics Lab, Research Program on Biomedical Informatics (GRIB), IMIM Hospital del Mar Medical, Research Institute and Pompeu Fabra University, Barcelona, Spain; ²Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain

The difficulty to interpret the somatic variants constitutes a major hurdle to the identification of those genomic events more likely to be biologically and/or therapeutically relevant in each individual tumor. The information on genomic alterations that are validated as oncogenic and those that are found to influence the response to anti-cancer therapies is fragmented across the literature and several specialized resources. Moreover, there is no framework to automatically match the variants in an individual's tumor alterations to the available knowledge. Finally, most of the alterations observed in a tumor, even those occurring in cancer genes, remain of uncertain significance. Here we will present the Cancer Genome Interpreter (http://www.cancergenomeinterpreter.org), a resource that combines extensive expert curation and computational analyses to annotate the significance of alterations in tumor genomes according to distinct levels of relevance that respond to different use cases. The tool is aimed to support a broad range of applications in both pre-clinical and translational settings. As a proof-of-concept of its use, we will present the results of applying the tool to (a) systematically analyse 7000 tumors and reveal the landscape of genomic drivers and genomic-directed therapeutic opportunities—including potential repurposing opportunities— in cancer as it stands today; and (b) support the allocation of patients to the most appropriate clinical trial or to comprehensively explore off-label opportunities for genome guided therapies in patients unresponsive to standard-of-care treatment in two real-world clinical scenarios.
# S15: JAK.STAT SIGNALING: NEW TARGETS AND THERAPEUTICS FOR MULTIPLE DISEASES

## S15-1
INFLAMMATORY RE-TUNING OF IL-6 MEDIATED JAK-STAT SIGNALING

Jones S.A.

Division of Infection & Immunity, The School of Medicine, Cardiff University, Cardiff, Wales, UK

Interleukin (IL)-6 is recognized as major therapeutic target for the treatment of certain autoimmune diseases and cancer. Studies show that IL-6 has a broad effect on immune cells and structural stromal cells where it controls context-dependent pro- and anti-inflammatory activities. Clinical experience with IL-6 inhibitors has raised various questions about how and when to block this cytokine to improve disease outcome and patient benefit. Here, a better understanding of the receptor signaling events holds the potential to improve patient diagnosis, stratification and the choice of clinical intervention. To address this issue studies have considered the complexity of IL-6 receptor signaling in model systems. With a primary focus on IL-6 activities in inflammatory arthritis, studies will consider how cytokine control of the latent transcription factors Signal Transducer and Activator of Transcription (STAT)-1 and STAT3 affects immune outcomes. Attention will be given to IL-6 signaling in CD4 T-cells, and how T-cell activation re-tunes the signaling characteristics IL-6 to affect changes in T-cell proliferation, survival and effector function.

Funding: Arthritis Research UK Programme Grants (20770, 19796) Hoffmann la Roche Pharmaceuticals.

## S15-2
THE STAT3 PARADOX IN BREAST CANCER: AN INDUCER OF CELL DEATH AND AN ONCOGENE

Watson C.J.

Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, UK

STAT3 is well established as an oncogene and is frequently constitutively activated in breast cancers. Established breast cancer cell lines with high levels of STAT3 phosphorylation often exhibit addiction to STAT3 and undergo cell death when STAT3 is inhibited or ablated. Conversely, Stat3 is specifically activated in mouse mammary gland at the onset of post-lactational regression (involution) and conditional deletion of Stat3 in mammary epithelial cells abrogates cell death and delays involution. We have demonstrated that Stat3 induces a lysosomal pathway of cell death and switches cell fate from milk secretion to a phagocytic function. We are currently investigating the mechanism by which Stat3 mediates these effects. Given the challenges of developing drugs that specifically target STAT3, we anticipate that insights gained from these studies will be informative for the design of therapeutics that target essential components of the STAT3 pathway.

## S15-3
JAK-STAT PATHWAY IN MYELOPROLIFERATIVE NEOPLASMS: FROM JAK2 V617F TO CALRETICULIN MUTANTS

Constantinescu S.N.

Ludwig Cancer Research Brussels and Université catholique de Louvain, de Duve Institute, Brussels, Belgium

Myeloproliferative neoplasms (MPN) are diseases of the hematopoietic system whereby hematopoietic stem cells acquire mutations that give a major clonal advantage to myeloid progenitors leading excessive production of functional red blood cells, platelets and granulocytes. MPNs can accelerate and transform into acute myeloid leukemia, which is very difficult to treat. The most prevalent MPNs are Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis (MF), where patients suffer from excessive red blood cell, platelet and scarring of the marrow, respectively. Together PV, ET and MF are five times more prevalent than chronic myeloid leukemia induced by BCR-ABL. The molecular basis of MPNs is represented by acquired mutations that lead to persistent, non-regulated activated of JAK2 and the pathways downstream, STAT5/STAT3/STAT1, MAP-kinase and PI-3'-kinase. 70% of MPN carry the unique acquired somatic JAK2 V617F mutation, where a pseudokinase domain mutation removes inhibitory effects of pseudokinase domain on kinase domain, and also, activates a circuit via the SH2-JH2 linker to the kinase domain. We identified the first conformational event, formation of the aromatic stacking F595-F617 interaction, which triggers an activating circuit to the kinase domain. This circuit is conserved in JAK1 and TYK2, where homologous of V617F activate in a similar manner. The next somatic mutation playing a driver role is represented by W515L/K/A mutations in the thrombopoietin receptor (TpoR), which are present only in ET and MF (3-7%, respectively). We have identified the W515A mutation as being active, and showed that the W515 is part of an inhibitory amphipathic helix, that is required to maintain TpoR inactive. In the case of TpoR W515 mutations, it is JAK2 wild type that is pathologically activated. Importantly, we have also shown that 17 out of the 20 amino acids can activate at TpoR W515, thus a complete sequencing will be necessary. At the end of 2013, 20-35% of ET and MF cases were denoted double negative as they did not carry JAK2 V617F or TpoR mutations. The groups of R. Kralovics and T. Green discovered mutations in the calreticulin gene uniquely in ET and MF (15% and 25%, respectively). We have examined the mechanism of MPN induction by these CALR mutations, where deletions and insertions in exon 9 shift the frame by +1, resulting invariably in the same novel C-terminal positively charged tail, where the natural KDEL endoplasmic reticulum retention sequence is deleted. We have then shown in collaboration with the Kralovics and Vainchenker laboratories that CALR mutants exhibit the ability to activate the TpoR, by binding to its extracellular domain, promoting traffic in an immaturely N-glycosylated form through the secretory pathway to the cell surface. Thus, CALR mutants activate constitutively TpoR, and this pathologic signaling can be inhibited by JAK2 inhibitors. On the other hand, careful signaling experiments in cell lines and treatment with combinations of inhibitors show that JAK2 and MAP-kinase are key for CALR mutant effects, while for JAK2 V617F, JAK2 and PI-3'-kinase were key for transformation. Since mutant CALR proteins activate TpoR signaling from intracellular compartments and from the cell-surface, the next challenge is to assess the different contributions of these localizations to the overall phenotype. In summary, the transforming effects of CALR mutants represents a novel oncogenic mechanism, whereby a mutated chaperone activates its client and induces pathologic traffic.
S15-4

TARGETING STAT5 IN LEUKEMOGENESIS – WHAT'S
NEW?

Sexl V.

Institute of Pharmacology, Veterinary University of Vienna, Austria

JAK-STAT signalling has been identified as one of the core cancer
pathways. Mutations in JAK kinases are considered drivers of

malignancies, STAT transcription factors have been recognized as
important factors for tumor maintenance and progression. As a conse-
quence intensive efforts are ongoing to develop drugs targeting this
pathway. One of the key players in haematopoietic cancers is STAT5,
which is considered essential for several forms of leukemia. Novel
insights in the role of STAT5 in leukemia but also in NK cell medi-
ated tumor surveillance will be discussed.
POSTERS PRESENTATIONS

P-1
NEUROPROTECTIVE EFFECT OF NOVEL IMIDAZOLINE I₂ RECEPTOR LIGANDS IN THE SENESCENCE ACCELERATED MOUSE PRONE 8 (SAMP8)

Griñán-Ferré C.¹, Abás S.², Vasilopoulou F.¹, Erdozain A.M.³,
Keller B.⁴, Rodríguez-Arévalo S.², Callado L.F.³, García-Sevilla J.A.⁴,
Escolano C.², Pallás M.¹

¹Secció de Farmacologia, Institut de Neurociències, Barcelona, Spain;
²Laboratory of Medicinal Chemistry (Associated Unit to CSIC),
Facultat de Farmàcia i Ciències de l'Alimentació, Universitat de
Barcelona, Barcelona, Spain; ³Department of Pharmacology, Centro de
Investigacion Biomédica en Red de Salud Mental, CIBERSAM,
University of the Basque Country, UPV/EHU, Leioa, Bizkaia, Spain;
⁴Laboratory of Neuropharmacology, IUNICS/IdISPA, University of the
Balearic Islands (UIB), Palma de Mallorca, Spain

Cognitive impairment associated with age is characterized by changes
in neurons such as a reduction in the dendrite arborization and den-
dritic length, jointly with changes in a number of cell membrane recep-
tors. Some of these receptors have a prominent role in brain functions
as they are binding sites for the neurotransmitters modulating neurons'
responses. Recently, imidazoline I₂ receptors (I₂-IRs) have been sug-
gested as a potential therapeutic target for several CNS disorders such
as Alzheimer's disease (AD).

We have recently reported the pharmacological characterization of a
new family of (2-imidazoline-4-yl)phosphonates, prepared by a multi-
component microwaved-assisted reaction that fulfil the principles of
green chemistry. Radioligand binding studies showed that they dis-
played an outstanding affinity for I₂-IRs. Moreover, they showed also
neuroprotective effects.

In the present work, we present further studies on the neuroprotective
properties of these compounds. To this end, 12 months-old SAMP8
female mice were distributed in three groups: Control (n = 10), com-
pound 1 (n = 8), and compound 2 (n = 8). Mice were treated for
4 weeks with compounds 1 and 2 at the dose of 5 mg/kg/day added to
drinking water.

Both compounds induced a significant increase in motor activity, mea-
sured by open field, and cognitive improvement, through novel object
test. These changes were accompanied by a decrease in Tau hyper-
phosphorylation and amyloid precursor protein (APP) processing. A
significant Bax and Calpain protein level diminution, and Bcl-2
increase, indicated modifications in apoptosis process. Finally, qPCR
measured gene expression of *IL-6*, *IL-1β* and *Cxcl10* inflammation
markers as well as the oxidative stress markers *Tnf-α*, *iNOS*, *Hmox1*
and *Aldh2* were reduced in the treated mice compared to the control
groups. Altogether these results demonstrated the neuroprotective
effect of these novel I₂-IRs ligands in a neurodegenerative disease ani-
mal model of AD.

**Keywords:** Neurodegeneration, (2-imidazoline-4-yl)phosphonates, imi-
dazoline receptors I₂, aging.

**Acknowledgments:** This study was supported by the Ministerio de Economía y Competitividad and Fondo Europeo de Desarrollo Regional (MINECO-FEDER) (Projects BFU2015-66030-R and SAF2016-77703-C2-1-R)

P-2
CHRONIC Δ⁹-THC ADMINISTRATION LEADS TO FUNCTIONAL 5HT2A RECEPTORS SUPERSENSITIVITY MEDIATED BY INHIBITORY G/I/O PROTEINS IN MOUSE BRAIN CORTEX

Ibarra-Lecue I., Mollinedo-Gajate I., Meana J.J., Callado L.F.,
Diez-Alarcia R., Urigüen L.

Department of Pharmacology, University of the Basque Country,
UPV/EHU, Leioa and Centro de Investigación Biomédica en Red de
Salud Mental (CIBERSAM), Leioa, Spain

Several findings suggest that 5HT2A receptors (5HT2AR) may be
involved in the molecular mechanisms responsible for psychotic
symptoms. Hallucinogenic drugs acting as 5HT2AR agonists, such as
psilocybin, lysergic acid diethylamide (LSD) and dimethoxyiodoam-
phetamine (DOI) produce psychosis-like symptoms in healthy subjects.
On the other hand, cannabis intoxication induces in healthy subjects
cognitive alterations similar to those seen in schizophrenia and worsens
the symptoms in schizophrenic patients. These data may suggest a link
between cannabis and psychosis. Our aim was to investigate the status
and functionality of 5HT2AR in brain cortex of adolescent mice chron-
ically treated with Δ⁹-THC.

Mice were treated during adolescent period with Δ⁹-THC (10 mg/kg daily, 30 days, i.p.) or vehicle. Prepulse inhibition test (PPI) was performed in both groups at basal conditions and after acute DOI injection (0.5 mg/kg, i.p.). Displacement curves of specific [³H]ketanserin binding (2 nM) by DOI (10⁻¹² to 10⁻³ M) were also carried out in brain cortex membranes. Moreover, specific stimulation of different Gα proteins by DOI (10⁻⁵ M) following a [³⁵S]GTPγS binding assay combined with immunoprecipitation was determined.

Chronic Δ⁹-THC significantly potentiated (p < 0.05) the DOI-induced reduction in the PPI. Additionally, although no changes were found in the density of 5HT2AR, a significant increase (p < 0.01) in the affinity of G-protein pre-coupled population of 5HT2AR was observed in brain cortex of Δ⁹-THC-treated mice. Moreover, a significant increase (p < 0.05) in the 5HT2AR-mediated stimulation of G<sub>αi1</sub>, G<sub>αi3</sub>, G<sub>αi0</sub> and G<sub>αz</sub> protein subtypes but not of G<sub>αs</sub> or G<sub>αq/11</sub> subunits was found in Δ⁹-THC-treated mice compared to controls.

Our results show that, in mice brain cortex, chronic exposure to Δ⁹-
THC during adolescence enhances the 5HT2AR ability to activate
specific pathways of inhibitory G<sub>α</sub> protein subtypes. This finding sug-
gests that chronic cannabis exposure induces a pro-hallucinogenic
5HT2AR conformation that may facilitate the development of psy-
chosis.

**Key words:** 5HT2AR, Δ⁹-THC, mouse brain.
P-3

IDENTIFICATION OF PHARMACOLOGICAL CHAPERONES FOR THE CELLULAR PRION PROTEIN BY DYNAMIC MASS REDISTRIBUTION SCREENING

Iglesias A.¹, Elezgarai S.R.²,³, Barreca M.L.⁴, Moreno J.⁵, García S.⁵, Castilla J.⁵,⁶, Loza M.¹, Biasini E.³,⁷, Domínguez E.¹, Requena J.R.⁸

¹BioFarma Group, CIMUS Biomedical Research Institute, University of Santiago de Compostela, Spain; ²Department of Molecular Biochemistry and Pharmacology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy; ³Dulbecco Telethon Institute, Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy; ⁴Dipartimento di Scienze Farmaceutiche, Università degli Studi di Perugia, Perugia, Italy; ⁵CIC bioGUNE, Parque tecnológico de Bizkaia, Bizkaia, Spain; ⁶IKERBASQUE, Basque Foundation for Science, Bilbao, Bizkaia, Spain; ⁷Department of Neuroscience, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy; ⁸CIMUS Biomedical Research Institute, University of Santiago de Compostela-IDIS, Spain

Genetic prion diseases are dominantly inherited neurodegenerative disorders linked to mutations in the *PRNP* gene encoding the prion protein (PrP). One of these mutations, D178N, is likely to be causal¹ and may trigger the conversion of the cellular prion protein (PrP<sup>C</sup>) into an aggregated form (PrP<sup>Sc</sup>) that self-propagates in the brain by imposing its abnormal conformation². This concept provides a rationale for tackling aggregation by stabilizing the PrP<sup>C</sup> monomeric protein precursors by increasing the Gibbs free energy barrier (ΔG) required for the initial misfolding events. This goal could potentially be achieved with ligands of PrP<sup>C</sup> that act as pharmacological chaperones. In order to identify such compounds, we developed a novel screening method based on Dynamic Mass Redistribution (DMR), a label-free, fully automated biophysical technique, capable of detecting small molecule-protein interactions. Two High Throughput Screening (HTS) campaigns were carried out, first with Prestwick® chemical library (1280 compounds) and second with a diversity library (3888 compounds). The diversity library was designed selecting compounds with an optimal molecular weight to bind with PrP<sup>C</sup> and which cross the blood-brain barrier.

Active compounds were selected for cherry picking and triaged by Protein Misfolding Cyclic Amplification assay³, a technique that mimics prion replication in vitro. DMR-based HTS campaign identified 13 active compounds in Prestwick® chemical library which stabilize the conformation of human PrP<sup>C</sup>. CBG030937 stands out from others for its high affinity. Moreover, it inhibits in a dose-dependent manner the conversion of PrP<sup>C</sup> into PrP<sup>Sc</sup>. HTS of diversity library is ongoing.

Our results demonstrated that DMR is a reliable platform for the identification of novel pharmacological chaperones targeting the cellular prion protein.

1. Minikel, E. V. et al. *Sci. Transl. Med.* 8, 322ra9 (2016)

2. Vázquez-Fernández, E. *et al. Plos Pathog.* 12 (9) 1 (2016)

3. Saborio, G. P. et al. *Nature.* 411, 810–813 (2001)

P-4

GENDER DIFFERENCES ON THE EFFECTS OF PRENATAL STRESS AND PERINATAL DIET ON THE BEHAVIORAL PROFILE OF AGED RATS

Bengoetxea X.¹, Paternain L.², Martisova E.¹, Milagro F.I.²,³,
Martínez J.A.²,³,⁴, Campión J.²,³, Ramirez M.J.¹,⁴

¹Department of Pharmacology and Toxicology, University of Navarra,
Pamplona, Spain; ²Department of Nutrition, Food Science and
Physiology, Centre for Nutrition Research, University of Navarra,
Pamplona, Spain; ³CIBERobn, Centro de Investigación Biomédica en
Red de la Fisiopatología de la Obesidad y Nutrición, Carlos III Health
Institute, Madrid, Spain; ⁴IDISNA, Navarra's Health Research
Institute, Pamplona, Spain

Epidemiological studies have provided compelling evidence that chronic exposure to stress is associated with significantly increased risk of developing psychiatric disorders in adulthood, such as depression or schizophrenia. Gender is one major variable that appears to confer differential vulnerability to stress. On the other hand, previous studies have reported nutrition as a factor that can modify the stress response and even more, maternal nutrition, could also influence fetal programming. The present work investigates whether chronic perinatal stress (PS) influences the long-term sex-dependent neuropsychological status of offspring and the effects of an early dietary intervention in the dam. To this end, dams were fed either a high fat sugar diet (HFSD) or methyl donor supplementation diet (MDSD). PS procedure did not affect body weight of the offspring. MDSD induced decreases in body weight both in male and female offspring (1 month) that were still present in aged rats. HFSD induced an increase in body weight both in male and female offspring that did not persist in aged rats. In the Porsolt forced swimming test, only young PS males showed depressive-like behavior. This increase in immobility time was reversed by MDSD. In both old male and female rats (20 months), PS induced cognitive impairment in the Morris Water Maze that was reversed by MDSD. HFSD induced cognitive impairment in both control and PS old rats, but there was no additive effect of PS and HFSD. It is proposed here that the diversity of symptoms following PS could arise from programming effects in early brain development and that these effects could be modified by the dietary intake of the dam.

**Key words:** chronic perinatal stress; methyl donor supplemented diet; high fat sugar diet; cognition.

P-5

EXOCYTOSIS AND MITOCHONDRIAL ULTRASTRUCTURE AND FUNCTION IN CHROMAFFIN CELLS OF THE SOD1<sup>G93A</sup> MOUSE MODEL OF ALS AT A PRE-DISEASE STAGE

Méndez-López I., Martínez-Ramírez C., Prieto Simancas P., Ronda del Corro M., Sáez Albendea L., Villaverde Rebenaque P., García A.G., Padín Nogueira J.F.

Instituto Teófilo Hernando and Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain

Paralysis and respiratory failure in patients of amyotrophic lateral sclerosis (ALS) are due to the selective loss of motor neurons. Excess synaptic glutamate release and Ca²⁺-dependent excitotoxicity has been hypothesized to be involved in ALS pathogenesis. We recently found that in chromaffin cells of a SOD1<sup>G93A</sup> mouse model of familiar ALS, the fusion pore kinetics and cell excitability are diminished at postnatal age P100-120, when the disease is already established (Calvo-Gallardo et al., Am J Physiol Cell Physiol 2015;308:C1-C19). Here we have investigated whether exocytosis and mitochondrial ultrastructure are altered at early disease stages (P30-P40) in CCs of mouse SOD1<sup>G93A</sup>. Transmission electron microscopy indicated that with respect to wild-type adrenal medullary CCs, mitochondria from SOD1<sup>G93A</sup> CCs showed the following alterations: 1, more number and small sized; 2, increased mitochondrial intermembrane space; 3, swollen cristae. These ultrastructural changes were accompanied by lower ATP levels and a higher rate of reactive oxygen species (ROS) generation. In spite of the low ATP, the kinetics of the exocytotic fusion pore SOD1G93A CCs have not been dramatically changed, as we observed once the paralysis is already established in P100-120 mice. This suggests that ultrastructural alterations and mitochondrial dysfunction in SOD1<sup>G93A</sup> occurring at early predisease stages (P30-P40), have lower physiological relevance than in later stages, but these results could generate some clues about the initiation and progression of the disease.

**Key words:** Amyotrophic Lateral Sclerosis; SOD1<sup>G93A</sup>; mitochondrial ultrastructure; chromaffin cell.
P-6
DOPAMINERGIC DEGENERATION PARTIALLY MODIFIES
THE RESPONSE OF THE LOCUS COERULEUS TO NOXIOUS
STIMULUS

Paredes-Rodriguez E., Ugedo L., Morera-Herreras T., Miguélez C.

Department of Pharmacology, Faculty of Medicine and Nursing,
University of the Basque Country, Leioa, Bizkaia, Spain

Pain is a major complaint for up to 85% of Parkinson's disease (PD) patients; however, its pathophysiology is still poorly understood. There is an increasing evidence suggesting that the noradrenergic system, in particular the locus coeruleus (LC) nucleus, is directly related to the development of pain. Given that previous studies have demonstrated that PD is associated with dysfunctional neuronal activity of the LC, we examined the implication of this nucleus in pain modulation in a rat model of PD.

The aim of the present study was to investigate if LC response to nox-
ious stimulus was modified in 6-hydroxydopamine (6-OHDA) lesioned
rats. To do that, single-unit extracellular recordings were carried out
under chloral hydrate anesthesia in sham and 6-OHDA lesioned rats
before and after noxious stimulus (ipsilateral and contralateral paw
compression). Neuronal activity at baseline and parameters of noxious
stimulus-evoked response were analyzed. In a set of experiments L-
DOPA effect was also analyzed.

Dopaminergic denervation did not modify the spontaneous activity or
the noxious stimulus-evoked discharge parameters (duration, suppres-
sion period and frequency) of LC neurons. However, the incidence of
the noxious stimulus-evoked response in LC neurons after contralateral
paw compression was significantly lower in 6-OHDA lesioned rats
compared to that obtained in sham rats. L-DOPA administration
(24 mg/kg, plus benserazide 15 mg/kg; intraperitoneal) decreased sig-
nificantly the incidence of the noxious stimulus-evoked response in
LC neurons after paw compression in 6-OHDA lesioned and sham
rats.

This work provides evidence that the LC-noradrenergic system is
involved in the alteration of pain modulation after dopaminergic dener-
vation. Both, 6-OHDA lesion and L-DOPA exogenous administration
modify the incidence of neuronal response to noxious stimulus.
Supported by SAF2016-77758-R (AEI/FEDER, UE) and IT 747-13.
Elena Paredes-Rodriguez has a predoctoral fellowship for the Training
of Personal Research from the Basque Government.

**Key words:** locus coeruleus, 6-OHDA lesion, Parkinson's disease, pain

P-7
BRAIN SPECIFIC CARNITINE PALMITOYLTRANSFERASE 1C (CPT1C) IS INVOLVED IN THE HYPOTHALAMIC REGULATION OF THERMOGENESIS BY ENDOCANNABINOIDS-DEPENDENT MECHANISMS

Rodríguez-Rodríguez R.¹, Miralpeix C.¹, Pozo M.¹, Casals N.¹,²

¹Basic Sciences Department, Faculty of Medicine and Health Sciences, Universitat Internacional de Catalunya, Sant Cugat del Vallés, Barcelona, Spain; ²CIBER Fisiopatología de la Obesidad y la Nutrición (CIBEROBN), Instituto de Salud Carlos III, Madrid, Spain

Brain-specific carnitine palmitoyltransferase-1C (CPT1C), despite its
minimal CPT1 catalytic activity, is implicated in central regulation of
food intake and energy homeostasis. CPT1C knock-out (KO) mice
show an increased susceptibility to diet-induced obesity and an
impaired regulation of food intake in response to leptin or ghrelin.
However, it is completely unknown whether CPT1C is involved in
hypothalamic regulation of thermogenesis. Here we explore the role of
CPT1C in leptin- and diet-induced thermogenesis in brown adipose tis-
sue (BAT) and the mechanisms involved.

CPT1C KO and WT mice were fed a standard or high fat diet (HFD) for 7 days. In WT mice, HFD significantly increased interscapular BAT (iBAT) temperature and thermogenic markers expression, while

these parameters were lower in CPT1C KO mice. Also, KO animals evidenced higher body weight gain and adiposity as well as hyperleptinemia, suggesting an earlier onset of leptin resistance. 4 h-Central leptin injection induced a sustained increase of iBAT temperature and upregulation of thermogenic markers in WT mice, whereas these responses were significantly attenuated in KO mice. Phenotype reversal studies identified the mediobasal hypothalamus (MBH) as the area of CPT1C activity responsible for those effects.

Evaluation of the mechanisms involved revealed a dysregulation of leptin signaling markers (i.e. p-AMPK, p-STAT3 and SOCS3) in MBH from CPT1C KO mice compared to WT mice. KO mice also showed increased hypothalamic levels of the endocannabinoid 2-arachidonoylglycerol (2-AG), which is associated to an obesogenic phenotype. In relation to this, proteomic and FRET analysis confirmed the interaction of CPT1C and ABHD6, which is a 2-AG hydrolase involved in the hypothalamic regulation of thermogenesis. Therefore, our data demonstrate that CPT1C is crucial in the hypothalamic regulation of BAT thermogenesis by leptin signaling and 2-AG-dependent mechanisms in the MBH and suggests CPT1C as a potential central target to fight against obesity and its metabolic complications.

P-8
REPEATED ELECTROCONVULSIVE SHOCK INCREASED
NEWLY BORN GENERATED CELLS AND
NEUROPLASTICITY MARKERS IN THE RAT
HIPPOCAMPUS

García-Cabrerizo R., García-Sevilla J.A., García-Fuster M.J.

IUNICS, University of the Balearic Islands, and IdISBa, Palma de Mallorca, Spain

Electroconvulsive shock (ECS) is a widely used and effective treatment in psychiatry, especially in major depressive disorders, but the basis for its therapeutic effect is still unknown. Thus, the aim of this study was to evaluate in male Sprague-Dawley rats the differential effects of a single (1 session) or repeated (5 sessions, 1 per day) exposure to ECS (95 mA, 0.6 s, 100 Hz, via earclip electrodes) on hippocampal plasticity markers. Earclip electrodes were also applied to control rats but without electrical current (sham-control). Rats were sacrificed 24 h after the last ECS session by decapitation. The left hippocampus was cryostat-cut and slide-mounted for ascertaining cell genesis markers (i.e., Ki-67 for recent cell proliferation and NeuroD for immature neurons) by immunohistochemistry and the right hippocampus was freshly dissected for studying neuroplasticity markers (i.e., BDNF, FADD and p-FADD) by western blot analyses. The main results showed that repeated ECS (vs. sham-controls) increased hippocampal: (1) cell proliferation (Ki-67 positive: (+464 ± 34%, *p* < 0.001); (2) mature BDNF protein content (+618 ± 80%, *p* < 0.001), which is associated with neuronal survival, growth and diferentiation; and (3) p-FADD protein content (59 ± 25%, *p* < 0.05), which is considered a neuroplasticity/anti-apoptotic marker. Taken together, the enhanced cell proliferation, accompanied by the upregulation of BDNF and p-FADD contents, indicate neuroplastic effects of repeated ECS in the rat hippocampus. NeuroD immunohistochemistry is currently being analyzed and ongoing immunofluorescence experiments are being carried out to assess the possible association (co-localization) of these neuroplasticity markers with particular stages of cell genesis. Supported by ‘Delegación del Gobierno para el Plan Nacional sobre Drogas’ (MSSSI, Grant 2016/002), ‘Ramón y Cajal’ Program to M.J.G.-F. (MINECO) and RTA-RD16/0017/0010.

**Key words:** electroshock; rat; hippocampal neurogenesis; neural plasticity
P-9
NOVEL COUMARIN DERIVATIVES AS SELECTIVE MAO-B INHIBITORS: *IN VITRO* AND *IN VIVO* ASSAYS

Rodríguez-Enríquez F., Quezada E., Matos M.J., Costas-Lago M.C., Mayán L., Laguna R., Uriarte E., Viña D., Fontenla J.A.

Pharmacology of Chronic Diseases, Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidade de Santiago de Compostela, Santiago de Compostela, Spain

As a consequence of an increasingly aging population, the number of people affected by neurodegenerative disorders is rapidly increasing. Although the etiology of these diseases has not been completely defined, common molecular mechanisms including neuroinflammation, excitotoxicity, oxidative stress, mitochondrial dysfunction and deficits in neurotransmitters have been confirmed and can be targeted therapeutically. Among these disorders, Parkinson's disease (PD) is one of the most prevalent characterized by degeneration of nigrostriatal dopaminergic neurons, aggregation of α-synuclein and motor symptoms¹.

Current dopamine-replacement strategies provide symptomatic relief, however their effectiveness wear off over time and their prolonged use leads to disabling side-effects in PD patients. There is therefore a critical need to develop new drugs and drug targets to protect dopaminergic neurons from degeneration in PD².

In this research, natural or synthetic coumarins appropriately substituted have shown interesting MAO inhibitory activity³. In recent years, our group have evaluated more than one thousand coumarin derivatives and confirmed that substitution at 3 or 7 position leads to potent

and selective *in vitro* hMAO-B inhibitors⁴.

Our next goal is to know the *in vivo* possibilities of these derivatives. Therefore, we have selected three new molecules which exhibited a promising *IC*₅₀ hMAO-B (1: 1.05 µM; 2: 0.246 µM; 3: 0.312 µM) as well as adequate theoretical ADME-related physicochemical/structural parameters (miLogP 1: 3.275; 2: 3.518; 3: 3.935) for *in vivo* administration. Moreover, *in vivo* assays have shown that none of these coumarin derivatives exhibits toxicity in a 100 mg/Kg intraperitoneal administration in mice and all the compounds significantly modify parameters studied: speed, % movement and distance moved.

[1] Aarsland D. Nat Rev Neurol. 2017 Mar 3. https://doi.org/10.1038/nrneurol.2017.27

[2] Santangelo G. Neurosci Biobehav Rev. 2017 Feb 24. pii: S0149-7634(16)30434-1.

[3] Skalicka-Woźniak K. Pharmacol Res. 2016;103:188-203.

[4] Matos MJ. Curr. Top Med Chem. 2012;12:2210-39.

**Key words:** Neurodegenerative disease; Parkinson's disease; coumarin; MAO-B inhibitors

P-10
GENE-BASED ASSOCIATION ANALYSIS OF GENE NETWORKS FOR THE STUDY OF EXTRAPYRAMIDAL SYMPTOMS INDUCED BY ANTIPSYCHOTICS

Boloc D., García-Cerro S., Gassó P., Gortat A., Rodríguez N., Torres T., Martínez A., Lafuente A., Bernardo M., Mas S.

Department of Pathological Anatomy, Pharmacology and Microbiology, University of Barcelona, Barcelona, Spain; institut Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Madrid, Spain

**Introduction:** Acute extrapyramidal symptoms (EPS) induced by antipsychotic (AP) treatment are frequent and serious adverse reactions of AP drugs. Pharmacogenetic studies identified several associations between EPS and genetic markers in candidate genes, however, these associations did not explained the whole variability of the EPS. In the present study, we applied an integrative approach to overcome some of the limitations detected in pharmacogenetic studies of EPS.

**Material and Methods:** Microarrays data from different models will be integrated to create a protein-protein interaction (PPI) network that will represents the molecular signature of AP induced EPS. The network will be analyzed to identify key components. SNPs in regulatory elements of these genes will be genotyped. Finally, gene-based algorithms will be applied in a discovery sample (243 chronic psychiatric patients) and replicated in an independent sample (109 first episode psychotic patients).

**Results:** The integration of omics data allowed the construction of a PPI network with 17.868 nodes, of whom 1.368 nodes where common to all models and served as a base for the network analysis. After PPI network was analyzed 28 candidate genes were selected, and 137 SNPs in regulatory elements of these genes were genotyped. Gene based analysis in the discovery sample identified nine genes associated with EPS (OAS1, ADARB2, DMD, ARPP21, KALRN, SHROOM3, MAST4, PINX1, ABL1), and four where consistency replicated in the replication sample (OAS1, DMD, PINX1, ABL1).

**Discussion:** The replication of gene-based analysis in two independent population demonstrated the suitability of the strategy presented here to identify new candidate genes for pharmacogenetic studies of EPS. Genes associated to EPS where involved in cytoskeleton regulation (DMD, ABL1) and RNA editing (OAS1, ABL1), and where previously associated to movement disorders, such as parkinsonism and muscular dystrophy, and also to metabolic disorders, such as diabetes mellitus type 1 and rheumatoid arthritis.

P-11
THE EFFECT OF BUSPIRONE ON THE BASAL GANGLIA OUTPUT NUCLEI IS RELATED TO THE DOPAMINERGIC SYSTEM INTEGRITY IN RATS

Vegas-Suárez S., Miguélez C., Morera-Herreras T., Ugedo L.

Department of Pharmacology, Faculty of Medicine and Nursing, University of the Basque Country (UPV/EHU), Leioa, Bizkaia, Spain

The basal ganglia (BG) nuclei form an integrative subcortical network directly related with the motor performance. The pathophysiology of Parkinson's disease (PD) and L-DOPA-induced dyskinesia (LID) is associated with dysfunctional neuronal activity and changes in the oscillatory activity and synchronization of the cortical-BG-thalamus circuits. Serotonin-based therapies have promising results in the treatment of PD and LID. Interestingly, buspirone, a partial agonist of 5-HT₁A receptors, has shown antidyskinetic properties in humans and different animal models of PD, but the mechanisms involved in this therapeutic effect are not fully understood.

The aim of the present study was to investigate the effect of buspirone on the neuronal activity of the BG output nuclei; the substantia nigra pars reticula (SNr) and entopeduncular nucleus (EP). To do that, single-unit extracellular recordings were carried under urethane anaesthesia in control and 6-hydroxydopamine (6-OHDA) lesioned rats. Neuronal activity parameters, oscillatory activity and synchronization between these nuclei and the motor cortex were analyzed.

In the EP, local (0.25–2 nmol) and systemic (0.625–5 mg/kg i.v.) administration of buspirone consistently reduced neuron activity in control and 6-OHDA lesioned rats with a moderate denervation (between 60% and 80% of optical TH-immunoreactivity density). However, the systemic effect was absent in 6-OHDA lesioned rats with complete nigrostriatal denervation. The inhibitory effect was rapidly reversed by an antagonist of the 5-HT₁A receptors, WAY-100635 (1 mg/kg i.v.). In the SNr, local application of buspirone (0.25–2 nmol) also produced an inhibitory effect in lesioned, partially lesioned and control rats, but not when systemically (0.625–5 mg/kg i.v.) applied. Furthermore, the synchronization and low-frequency oscillatory activity in both nuclei or in the motor cortex remained
unaltered after drug application in 6-OHDA lesioned and control situations. Altogether, these results indicate that at least in the EP, buspirone produces an inhibitory effect, which is mediated by a complex mechanism that involves DA system as we had previously shown in the activity of the subthalamic nucleus.

*Supported by SAF2016-77758-R (AEI/FEDER, UE) and IT747-13
Sergio Vegas-Suárez has a predoctoral fellowship for the Training of Personal Research (UPV/EHU)*

**Key words:** 5-HT₁A receptors, 6-OHDA lesion, Electrophysiology recording, Oscillatory activity

## P-12
EFFECT OF ABCB1 C3435T POLYMORPHISM ON PHARMACOKINETICS OF ANTIPSYCHOTICS AND ANTIDEPRESSANTS

Saiz-Rodríguez M., Belmonte C., Cabaleiro T., López-Rodríguez R., Román M., Ochoa D., Talegón M., Abad-Santos F.

Clinical Pharmacology Service, Hospital Universitario de la Princesa, Instituto Teófilo Hernando, Universidad Autónoma de Madrid (UAM), Instituto de Investigación Sanitaria la Princesa (IP), Madrid, Spain

**Background:** P-glycoprotein, encoded by ABCB1 gene, is an ATP-dependent drug efflux pump which export substances outside the cell. The T allele of C3435T polymorphism located in this gene is frequently associated with a slower metabolizer phenotype, so homozygous T/T individuals could show higher plasma levels of drugs. The aim of the present study was to evaluate the effect of this polymorphism on pharmacokinetics (PK) of 4 antipsychotics (olanzapine, quetiapine, risperidone and aripiprazole) and 3 antidepressants (trazodone, sertraline and citalopram).

**Material and Methods:** Four hundred and forty-five healthy volunteers receiving a single oral dose of the mentioned drugs were genotyped by Real Time PCR for C3435T SNP. Plasma concentrations of the drugs were measured by high performance liquid chromatography with tandem mass spectrometry detection (LC/MS/MS). PK parameters were calculated using non-compartmental analysis. Differences in PK between individuals were analyzed by a parametric univariate analysis, ANOVA or T-test (to compare T allele carriers vs non-carriers).

**Results:** As shown in the following table, C3435T polymorphism had no significant effect on PK of olanzapine, quetiapine, sertraline or citalopram. As expected, T allele carriers showed a higher AUC and T₁/₂ for aripiprazole. Surprisingly, CI of risperidone and trazodone was higher in T allele carriers.

<table><thead><tr><th rowspan="3">Drug (n)</th><th colspan="6">ABCB1 genotype</th></tr><tr><th colspan="3">C/C</th><th colspan="3">C/T + T/T</th></tr><tr><th>AUC (ng·h/ml)</th><th>CI/F (ml/h·kg)</th><th>T₁/₂ (h)</th><th>AUC (ng·h/ml)</th><th>CI/F (ml/h·kg)</th><th>T₁/₂ (h)</th></tr></thead><tbody><tr><td>Olanzapine (61)</td><td>300.1</td><td>0.26</td><td>32.8</td><td>292.8</td><td>0.25</td><td>32.6</td></tr><tr><td>Quetiapine (79)</td><td>172.0</td><td>2.37</td><td>4.0</td><td>200.0</td><td>2.14</td><td>3.91</td></tr><tr><td>Risperidone (70)</td><td>64.1</td><td>0.64</td><td>6.9</td><td>34.3*</td><td>1.1*</td><td>3.6</td></tr><tr><td>Aripiprazole (148)</td><td>1514.8</td><td>66.62</td><td>47.1</td><td>1675.1*</td><td>62.12</td><td>54.4*</td></tr><tr><td>Trazodone (36)</td><td>9023.7</td><td>0.18</td><td>9.01</td><td>9876.1†</td><td>0.16†</td><td>9.46†</td></tr><tr><td>Sertraline (34)</td><td>924.8</td><td>1.98</td><td>26.05</td><td>794.8</td><td>1.93</td><td>24.24</td></tr><tr><td>Citalopram (17)</td><td>1142.5</td><td>0.38</td><td>32.8</td><td>1350.9</td><td>0.37</td><td>34.1</td></tr></tbody></table>

**Conclusions:** ABCB1 C3435T polymorphism can affect the pharmacokinetics in different ways, increasing the elimination in some drugs and reducing it in others.

## P-13
ENVIRONMENTAL ENRICHMENT IN THE ABSENCE OF PHYSICAL EXERCISE PRODUCES BENEFICIAL BEHAVIOURAL EFFECTS AND ANTI-OXIDATIVE EFFECTS IN RATS

Marmol F.¹, Chamizo V.D.², Sanchez J.¹, Torres M.N.²

¹Departament de Fonaments Clínicos, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain; ²Departament de Cognició, Desenvolupament i Psicologia de l'Educació, Facultat de Psicologia, Universitat de Barcelona, Barcelona, Spain

Early sensory experience is crucial on brain development and in the capacity for learning in adult life. It has been shown that both environmental enrichment (EE) and physical exercise can produce a variety of long term effects at neuroanatomical, neurochemical, and behavioural levels in different species. In a recent study with rats (Chamizo et al., 2016) the animals had been reared in either EE plus wheel running or control conditions. The results revealed beneficial effects of both EE and wheel running: EE rats learned more and showed less anxiety than controls. However, an undeniable weakness of this previous study is that there is not a clear distinction between EE and physical exercise (i.e. voluntary wheel-running). The present work, with male rats, addresses this problem. After weaning, the rats were housed in pairs in enriched or in standard cages, for two and a half months in the absence of wheel running. When the rats were approximately 90 days old they were tested in the Morris pool (as in Rodríguez et al., 2010). EE rats reached the platform faster than control animals and performed better on a subsequent test trial without platform with the geometry cue individually presented. Behavioural anxiety was also measured by means of thigmotaxis. Then, the rats were sacrificed and the hippocampus was extracted (Marmol et al., 2015). Overall, the measures taken in the hippocampus revealed that EE rats showed higher values for antioxidant measures (total radical antioxidant parameter and catalase levels) and lower values for oxidative stress parameters (TBARS, protein oxidation activity and superoxide anion levels) than control animals. In summary, our results reveal clear beneficial effects of EE in the absence of wheel running, both behaviourally and when subsequently testing oxidative stress parameters in the rat hippocampus.

**Keywords:** Environmental enrichment, Geometry learning, Landmark learning, Morris water maze, Thigmotaxis, Hippocampus of rats, Antioxidative measurements

## P-14
SYNTHETIC SULFATIDE-LIKE SULFOGLYCOLIPIDS AS POTENTIAL NEUROPROTECTORS IN BRAIN DAMAGE

García-Alvarado F.¹,², Fernández-Mayoralas A.³, García A.G.¹,²

¹Instituto Teófilo Hernando, Universidad Autónoma de Madrid, Madrid, Spain; ²Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain; ³Instituto de Química Orgánica General, CSIC, Madrid, Spain

Glycolipids are membrane lipids with an attached carbohydrate. Located predominantly at the outer leaflet of the plasma membrane, they contribute to cell recognition and membrane microdomain formation during differentiation, development, and immune reactions. Sulfation, phosphorylation and sialylation confer negative charge on glycolipids and such structures form a part of determinants for molecular interactions. We have synthesized sulfoglycolipid IG20 that has a structure very similar to sulfatides, a class of sulfated galactosylceramidase that are synthesized mainly in oligodendrocytes and belong to the great family of sphingosine derived sphingolipids. We have found that IG20 at 0.3–10 µM afforded neuroprotection in rat hippocampal slices stressed with veratridine, glutamate, or with oxygen plus glucose deprivation followed by reoxygenation (OGD/reox). The excess production of reactive oxygen species (ROS) elicited by those stressors was mitigated by IG20 that also restored the depressed levels of GSH and ATP and counteracted the augmented iNOS expression. Additionally, the

<footnote>*p < 0.05 comparing C/T plus T/T vs C/C.</footnote>

<footnote>†p < 0.05 comparing T/T vs C/T and C/C.</footnote>
neuroprotecting effects of IG20 were prevented in the presence of inhibitors of the signaling pathways JAK2/STAT3, MEK/ERK 1/2, and PI3k/Akt, consistent with its ability to augment the phosphorylation of JAK2, ERK1/2, and Akt. It seems therefore that the activation of phase II response and the Nrf2/ARE pathway are mediating the antioxidant and anti-inflammatory as well as the ensuing neuroprotective actions of IG20. We are now testing how relevant is the presence of the sulfate residue of IG20 for its properties here reported. To achieve this we have synthesized other three sulfatide-like glycolipids lacking sulfate or sulfoglycolipid lacking a double bond.

## P-15
### HDAC5 AND SIRT2: EPIGENETIC TARGETS FOR ANTIDEPRESSANT THERAPY

Irene M.-C.¹, Mercedes E.¹, Teresa D.-P.¹, Roberto C., Antonello M.², Clemens Z.², Sergio V.², Elena P.¹, *To Rosa M.*¹

¹Departamento de Farmacología y Toxicología (Universidad de Navarra); Instituto Superiore di Sanità, Rome

**Background and purpose:** Antidepressant action has been linked to increased synaptic plasticity in the prefrontal cortex (PFC), in which epigenetic mechanism could play a key role. Interestingly, we have recently identified the histone deacetylases enzymes HDAC5 and SIRT2 regulated by stress and antidepressant treatment. Here, we have studied comparatively, *in vivo* in the mouse PFC and *in vitro* in SH-SY5Y cells, the implication of these epigenetic targets in the molecular mechanisms of antidepressants.

**Experimental approach:** Mice (C57BL6, 8 weeks) were treated (21 days, i.p.) with imipramine (10 mg/kg), fluoxetine (15 mg/Kg) and reboxetine (15 mg/kg) or saline. In addition, SH-SY5Y cell cultures were incubated with these antidepressants (at 10 µM, 2 and 24 h) and with a novel selective HDAC5 inhibitor (MC3822, 5 µM, 24 h) presented here or a SIRT2 inhibitor (33i, 5 µM, 2 and 24 h). Epigenetic and synaptic plasticity markers where studied.

**Key results:** our study shows that antidepressants increased both *in vivo* and *in vitro* expression of the brain derived neurotrophic factor (BDNF), the vesicular glutamate transporter 1 (VGLUT1), and the acetylated histones 3 (AcH3) and 4 (AcH4). In addition, imipramine and reboxetine increased the phosphorylation form of HDAC5 (P-HDAC5), mediating cytoplasmic export of this enzyme. On the other hand, SIRT2 was downregulated by all antidepressants. Finally, specific inhibition of HDAC5 and SIRT2 with the compounds MC3822 and 33i respectively increased neuroplasticity markers *in vitro*.

**Conclusion and implications:** Here we present two epigenetic mechanisms, nucleocytoplasmic export of HDAC5 and SIRT2 downregulation, by which antidepressants could enhance synaptic plasticity markers linked to antidepressant action. Thus, it is highlighted the therapeutic potential of these epigenetic targets for major depression.

## P-16
### RESVERATROL PROTECTS C57BL/6J AGED MICE IN FRONT OF LPS ACUTE INFLAMMATORY INSULT

Palomera-Avalos V.¹, Griñán-Ferré C.¹, Sanfeliu C.², Pallàs M.¹, Canudas A.M.¹

¹Secció de Farmacologia, Institut de Neurociències and Facultat de Farmàcia i Ciències de l'Alimentació, Universitat de Barcelona, Barcelona, Spain; ²Institut d'Investigacions Biomèdiques de Barcelona (IIBB), CSIC, and IDIBAPS, Barcelona, Spain

**Introduction:** Neuroinflammation and oxidative stress (OS) are believed to play a pivotal role in development of cognitive impairment related with aging. Although there is no controversy that increase in OS in CNS is a negative event, the possible pleiotropic effect of proinflammatory cytokines such as IL-6 and TNF-α is unclear. There is evidence in both directions, positive and negative, because they have a

pleiotropic action as neuroprotective factors modulating synaptic plasticity, cell death and excitotoxicity. Lipopolysaccharide (LPS) has been widely used to study the acute inflammatory and OS response in brain, inducing proinflammatory cytokines and oxidative stress markers increases. Resveratrol is a polyphenolic compound found in grapes and other plants; it exhibits antioxidant, anti-inflammatory, anti-aging and although the protective role of resveratrol in the CNS is well established, the mechanisms of these effects are not fully understood.

**Aim:** We evaluate the effects of resveratrol on the inflammatory response and antioxidant defences in aged mice after inflammatory insult by LPS.

**Methods:** Males C57BL/6J mice 22 months-old were distributed in four groups and following 8 weeks of treatment: Control, Resveratrol, LPS, and LPS + Resveratrol. Control and LPS mice had access to standard chow and mice with resveratrol diet (control and LPS) had access to standard chow diet enriched with the polyphenol (1 g/Kg, w/ w). LPS mice were injected intraperitoneally (100 µg/Kg, i.p.) 3 h before sacrifice. In order to evaluate the effect of LPS we performed Western blot and Real Time PCR. Specific antibodies and primers were used to determine proteins and genes of interest.

**Results and conclusions:** The cytokines IL-6, TNF-α, IL1-β and Cxcl10 hippocampal gene expression were significantly increased in LPS-RV group. In the same way, iNOS, Hmox1, Aldh2 and Cox2 gene expression, antioxidant markers, were increased in LPS-RV group in reference to other groups. On the basis of our results the conclusions point to a possible increase of resilience of aged brain induced by resveratrol against LPS insult that can be useful in infection or any other situation that requires an activation of inflammatory defenses to fight against a possible harmful stimuli.

**Acknowledgments:** This study was supported by the Ministerio de Economía y Competitividad and Fondo Europeo de Desarrollo Regional (MINECO-FEDER) (SAF2016-77703-C2-1-R). Verónica Palomera-Avalos PhD scholarship (V/2014/216) Universidad de Guadalajara. México.

**Keywords:** Resveratrol, inflammation, hippocampus, oxidative stress, aging

## P-17
### PHARMACOLOGICAL MODULATION OF CALCIUM SIGNALLING AND EXOCYTOSIS IN CHROMAFFIN CELLS FROM C57BL6/J MICE

Martínez-Ramírez C., García A.G.

Instituto Teófilo Hernando y Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain

During cell activation, the variations in the concentrations of ionised Ca²⁺ in the cytosol ([Ca²⁺]ᵢ) determine the efficacy of pre-exocytotic and exocytotic steps during the process of hormone and neurotransmitter release. In adrenal chromaffin cells those responses are configurated by a functional triad formed by voltage-activated calcium channels (VACCs), the endoplasmic reticulum (ER), and mitochondria. The transfer of Ca²⁺ between these three compartments fine tunes the [Ca²⁺]ᵢ and catecholamine secretory responses during cell activation (Garcia et al., 2006, Physiol Rev. This Ca²⁺ circulation has been best studied in bovine and rat CCs. Scarce information is available in CCs from the mouse strain C57BL6/J (MCCs), that is being widely used as genetic carrier of mutations occurring in human disease. Therefore, here we have investigated how various drugs and manipulations that target the elements of the functional triad of MCCs, affect the quantal release of catecholamine monitored online at the single-cell level, with a carbon fibre microelectrode and amperometry. We have found that stimulation with physiological acetylcholine (ACh) triggers bursts of single-vesicle secretory spikes that have a kinetics considerable faster than the spikes evoked by other secretagogues. This could be due to differences in Ca²⁺ handling because when ACh was used in presence of mitochondrial protonophore FCCP that prevents the clearance of [Ca²⁺]ᵢ, the spike exhibited a slower kinetics. Also, the blockade by compound ITH1266 of the mitochondrial Na⁺-Ca²⁺ exchanger (mNCX) potentiated the inactivated ACh secretory response.
Furthermore, ER Ca²⁺ depletors such as SERCA blockers thapsigargin and cyclopiazonic acid, augmented secretion elicited by repeated ACh brief pulses. Hence, we conclude that ER-mitochondria Ca²⁺ exchange contributes to the regulation of the fine kinetics of exocytosis. These data create a frame for the extrapolation of the functional triad concept to transgenic mouse model of disease. We are presently testing this concept in transgenic mouse models of Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease.

## P-18
BLACK SWISS MICE: MOLECULAR ALTERATIONS RELATED TO MANIA-LIKE BEHAVIOUR

Montero E.C., Muñoz M.R., Blázquez P.S., Garzón J.

Neuropharmacology, Cajal Institute, CSIC, Madrid, Spain

Mania has long been recognized as aberrant behaviour indicative of mental illness which includes a variety of complex and multifaceted symptoms<fnref n="1" />. Even though the aetiology of the disease remains unwell defined, abnormal glutamatergic neurotransmission and dysregulation of GSK3β kinase, have been implicated in human mania². Recent work described that the Black Swiss mice from Taconic Farms (BStac) recreate various features of that observed in mania<fnref n="3" />. Also and in contrast with a putative control, Black Swiss mice from Charles River (BScr), BStac mice did not developed depressive-like behaviours after exposure to multiple forms of stress⁴. The goal of the present study was explore the potential molecular alterations in the manic BStac comparing to BScr mice. To this end we evaluate the levels of N-methyl-D-aspartate receptor (NMDAR) subunits and expression and phosphorylation levels of the Akt/GSK3β signalling pathway. The molecular exploration shows that neither NMDAR subunits not total GSK3β levels in the frontal cortex differ in both strain of mice. However, phosphorylation analysis revealed marked differences in the regulation of GSK3β, Then, we observed that Akt-mediated inhibitory phosphorylation of GSK3β at serine 9 (P-S9) decreased in BStac in comparison to BScr mice. Also, the activating phosphorylation of GSK3β at tyrosine 216 (P-Y216), was increased in BStac. Thus, our results indicate that BStac mice have manic-like behaviours typical of increased GSK3β function and inhibition of GSK3β attenuated the expression of pro-manic mechanisms allowing the expression of depressive-like behaviours. (Supported by Plan Nacional de Drogas 2014-012 and MINECO, SAF-2015-65420R)

1. Cassidy F. et al, 1998. Arch. Gen. Psychiatry 55, 27–32.

2. Jope R.S., Roh M.S., 2006. Curr. Drug Targets. 7, 1421–1434.

3. Juetten J., Einat H., 2012. Behav. Pharmacol. 23, 211–214.

4. Garzon J., et al, 2017. Scientific Reports 7:43468

## P-19
COMPUTATIONAL APPROACHES FOR THE UNDERSTANDING OF AGONIST EFFICACY IN STRUCTURALLY AND FUNCTIONALLY COMPLEX GPCRs

Giraldo J.<fnref n="1" />,², Dalton J.A.R.<fnref n="1" />, Lans I.<fnref n="1" />,<fnref n="3" />, Roche D.<fnref n="1" />,⁴, Zhou B.<fnref n="1" />, Bruzzese A.<fnref n="1" />, Ricarte A.<fnref n="1" />

The knowledge of GPCR activation mechanisms and the concomitant concept of agonist efficacy have evolved in the last years to encompass the avalanche of new findings and increasing complexity in pharmacology. A giant conceptual leap has been made from the original description of efficacy, as the property of agonists that produces a pharmacologic effect by the formation of a binary ligand-receptor

complex, to the present description, which includes multiple receptor pathways and multiple receptor binding sites. Moreover, the inherently complex properties of functional selectivity and allosterism are further complicated by the molecular interactions between the receptor and the diversity of molecules present in the membrane including water, ions, lipids and other receptors (homo and hetero-dimerization).

In an effort to explain and quantify agonist efficacy our group is performing a multiple approach to the understanding of receptor activation mechanisms which includes GPCR crystal structure analyses<fnref n="1" />,², molecular dynamics simulations<fnref n="3" />⁻⁵ and mathematical modeling⁶⁻⁸. In addition to published data<fnref n="1" />⁻⁸, new results on the stabilization of either the inactive or the active states of the β₂ adrenergic receptor depending on the charge distribution of membrane lipids, the relationship between functional selectivity and partial agonism for the 5HT₂A receptor and a new mathematical model for receptor heterodimerization are presented. Thus, we are fully convinced that the combination of molecular and mathematical approaches in an integrated framework may help to clarify the intricacies of the efficacy concept and to provide a mechanistic understanding of receptor function.

**References:**

1. J. A. Dalton, I. Lans, J. Giraldo, BMC.Bioinformatics. 16:124, https://doi.org/10.1186/s12859-015-0567-3 (2015).

2. I. Lans, J. A. Dalton, J. Giraldo, J Struct.Biol 192, 545–553 (2015).

3. J. A. Dalton, X. Gomez-Santacana, A. Llebaria, J. Giraldo, J Chem.Inf.Model. 54, 1476–1487 (2014).

4. I. Lans, J. A. Dalton, J. Giraldo, J Phys.Chem B 119, 9510–9519 (2015).

5. J. A. Dalton et al., Curr.Neuropharmacol. 14, 441–454 (2016).

6. D. Roche, D. Gil, J. Giraldo, Br.J.Pharmacol. 169, 1189–1202 (2013).

7. J. Giraldo, Trends Pharmacol.Sci 36, 1–2 (2015).

8. D. Roche, P. H. Van Der Graaf, J. Giraldo, Drug Discov.Today (2016).

**Acknowledgements:**

This work was supported by MINECO grants NEURON PCIN-2013-018-C03-02 and SAF2014-58396-R and Fundació La Marató de TV3 grant 110230.

## P-20
α₂A⁻ AND α₂C-ADRENOCEPTORS AS SIGNIFICANT TARGETS FOR DOPAMINE AND DOPAMINE RECEPTOR LIGANDS

Casadó-Anguera V.<fnref n="1" />, Sánchez-Soto M.<fnref n="1" />,², Moreno E.<fnref n="1" />, Bender J.<fnref n="3" />,⁴, Yano H.², Cai N.-S.², Meiler J.<fnref n="3" />,⁴, Cortés A.<fnref n="1" />, Ferré S.², Casadó V.<fnref n="1" />

¹Department of Biochemistry and Molecular Biomedicine, Faculty of Biology, University of Barcelona, and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), and Institute of Biomedicine of the University of Barcelona, Barcelona, Spain; ²Integrative Neurobiology Section, National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Baltimore, MD, USA; ³Department of Pharmacology, Vanderbilt University, Nashville, TN, USA; ⁴Center for Structural Biology, Vanderbilt University, Nashville, TN, USA

It has long been recognized that several brain areas show a mismatch between the innervation by the catecholamines norepinephrine and dopamine and the density of their canonical receptors. Particularly notorious is the low norepinephrine innervation and relative high density of Gi/o-coupled α₂A and α₂C adrenoceptors in the striatum, major target for the ascending dopaminergic system. This prompted the possibility that dopamine could provide an effective endogenous ligand for both adrenoceptors, but previous studies were inconclusive. In the present study, we addressed the same question by analyzing the ability of dopamine and several putative selective dopamine receptor ligands to bind and activate α₂A and α₂C adrenoceptors in transfected mammalian cells using sensitive bioluminescent resonance energy transfer-based techniques that detect ligand-dependent activation of specific G proteins or activation of their effector adenylate cyclase. Furthermore, we also analyzed their ability to bind to α₂ adrenoceptors in cortical

<footnote n="1">¹Institut de Neurociències and Unitat de Bioestadística, Universitat Autònoma de Barcelona, Bellaterra, Spain; ²Network Biomedical Research Center on Mental Health (CIBERSAM), Bellaterra, Spain;</footnote>

<footnote n="3">³Grupo de Bioquímica Teórica, Universidad Industrial de Santander, Bucaramanga, Colombia; ⁴Faculty of Economics and Social Sciences, Universitat Internacional de Catalunya, Barcelona, Spain</footnote>