methods that exploit publicly available knowledge bases are becoming a cost-effective approach to uncover unknown polypharmacology.²,³ PARP inhibitors are a new class of targeted small-molecule cancer therapeutics that have shown unexplained differential effects in cellular models and clinical trials.⁴ With two PARP inhibitors recently approved by the FDA for BRCA-mutated ovarian cancer and several more in late-stage clinical trials, it is essential to fully characterize their mechanism of action to help oncologists' decide which PARP inhibitor should be prescribed in each case. Here, we use computational target prediction and *in vitro* validation to identify new targets of PARP inhibitors that may explain their differential clinical effects. What appeared as a single robust class of PARP inhibitors with similar pharmacological properties should now be regarded as a set of PARP inhibitors with a different pharmacological profile that makes them essentially unique and thus expands largely their potential therapeutic opportunities in the framework of precision oncology.

**Keywords:** Systems pharmacology, precision oncology, PARP inhibitors, off-targets, polypharmacology, computational methods

**References:**

(1) Jalencas X, Mestres J. On the origins of drug polypharmacology. *Med. Chem. Commun.* 4, 80–87 (2013).

(2) Antolin AA, Workman P, Mestres J, Al-Lazikani, B. Polypharmacology in precision oncology: current applications and future prospects. *Curr Pharm Des, in press.*

(3) Tym J, Mitsopoulos C, Coker E, Razaz P, Schierz A, Antolin AA, Al-Lazikani B. canSAR: updated cancer research and drug discovery knowledgebase. *Nucleic Acids Res.* 44, D938–43 (2016).

(4) Antolin AA, Mestres J. Linking off-target kinase pharmacology to the different cellular effects observed among PARP inhibitors. *Oncotarget.* 5, 3023–28 (2014).

# P-96
## ACTINIC KERATOSIS: STANDARD COMPOUNDS VALIDATION IN BOTH *IN VITRO* AND *IN VIVO* MODELS

Armengol C.¹, Blanco A.¹, Carreño C.¹, Andrés M.², Pagán I.¹, Aniorte G.¹, Gavalà A.¹, Godessart N.¹, Balagué C.¹

¹Skin Biology and Pharmacology; ²Medicinal Chemistry & Screening, Almirall, R&D Centre, St. Feliu del Llobregat, Spain

Actinic keratosis (AK) is the most common premalignant skin tumor, affecting as many as 25% of adults in the northern hemisphere. It is due principally to long-term sun exposure and affects 50% of predisposed individuals more than 60 years of age in Europe. Without treatment, AK lesions may progress to squamous cell carcinoma (SCC) and invade the dermis. Depending on the nature of the growth and the patient's age and health, various treatment options are available. Isolated or widespread actinic keratosis lesions may be treated with topical formulations. In this regard, there are four topical active ingredients currently approved: 5-fluorouracil (5FU), diclofenac, ingenol mebutate and imiquimod, which represent distinct therapeutic classes. The goal of this work was to study the mechanism of action of these compounds in a battery of relevant cutaneous *in vitro* and *in vivo* assays which could provide measures of the anti-proliferative, cytotoxic or immunostimulant activity of those drugs. Different *in vitro* tests using HaCaT cells, normal human keratinocytes or human skin explants were used. In addition, the effect of the topical formulations was studied on mouse skin *in vivo* and on an isograft tumor model. AK reference standards show distinct behaviors in all the selected models, with 5-FU showing a marked antiproliferative activity and ingenol mebutate showing the strongest pro-inflammatory profile. Understanding these differences is key for the selection of new therapies for AK.

**Keywords:** Actinic keratosis; 5FU; ingenol; imiquimod; diclofenac; *in vitro* models; *in vivo* models

# P-97
## THE ROLE OF RECEPTOR PROTEIN TYROSINE PHOSPHATASE BETA/ZETA IN CELL MIGRATION

Pantazaka E., Castana P., Tassopoulos D., Poimenidi E., Papadimitriou E.

Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras

Receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) is highly expressed in human induced pluripotent and embryonic stem cells, while in adult humans, RPTPβ/ζ is mostly expressed in the brain, skin, stomach and testis. Interestingly, it is significantly over-expressed in the brain and lung of corresponding cancer patients compared with normal controls, suggesting that it may play a role in glioblastomas and/or lung cancer. We have previously shown that RPTPβ/ζ is a receptor for pleiotrophin (PTN) and vascular endothelial growth factor A (VEGF-A), mediating their stimulatory effect on human umbilical vein endothelial cell migration through interaction with α~v~β~3~ integrin. In the present work, we studied the role of RPTPβ/ζ in the effect of PTN and VEGF on the migration of microvascular endothelial cells, as well as glioblastoma cell lines that do not express α~v~β~3~. In all cases, RPTPβ/ζ has an effect on cell migration which is either stimulatory or inhibitory in a cell-dependent manner. In all cases, RPTPβ/ζ mediates the effect of PTN on cell migration, while the effect of VEGF-A seems to be affected in endothelial but not glioblastoma cells. Expression of α~v~β~3~ seems to explain, at least partly, the differential effect of RPTPβ/ζ on cell migration, although it seems that other factors may be also involved and are going to be discussed. Collectively, our data suggest that RPTPβ/ζ significantly affects cell migration and α~v~β~3~ is a key molecule in RPTPβ/ζ signaling. This knowledge, together with data on cancer cell characteristics and cancer angiogenesis, can lead to new therapeutic approaches in controlling brain and lung cancer development.

Acknowledgements: This research was partly supported by a Marie Curie Intra European Fellowship within the 7th European Community Framework Programme (ALTangioTARGET, grant agreement No 626057). Penelope Castana is a recipient of a PhD fellowship from the Hellenic State Scholarships Foundation (IKY).

**Keywords:** angiogenesis, cancer, tyrosine phosphatases, signaling

# P-98
## NIMOTUZUMAB AND RADIOTHERAPY IN THE TREATMENT OF PEDIATRIC PATIENTS WITH DIFFUSE INTRINSIC BRAIN ASTROCYTOMAS

Marrero Miragaya M.A.¹, Tirado Marrero M.², Gonzales Carrasco M.C.³, Crombet Ramos T.⁴, Palomino Machado L.¹

¹National Coordinating Center of Clinical Trials, La Habana, Cuba;

²'Hermanos Ameijeiras' Hospital, La Habana, Cuba; ³Oncology Department, 'Juan Manuel Marquez' Hospital La Habana, Cuba;

⁴Medical Division, Center of Molecular Immunology, La Habana, Cuba

A multicenter, phase II clinical trial to evaluate safety and efficacy of Nimotuzumab, a monoclonal antibody against epidermal growth factor receptor added to standard therapy for Brain Tumors in Pediatric Patients. Forty-five patients analyzed, 23 were included in Brazil and 22 patients in Cuba, since 2007 - 2012. There was a discontinuation of treatment in 38 patients, the progression of the disease being 68.4%. The efficacy analyzed in those patients who had received at least 12 doses of Nimotuzumab and 54 Gy or more of RT. A response observe in 20 patients (44.4%). The median progression-free survival and overall survival was equivalent to 9.03 and 15.1 months. There were no differences in overall or progression-free survival among patients treated in Cuba or Brazil. Progression-free survival at the sixth month was 72.9%. Five hundred sixty-nine, adverse events was reported, 178 of them (31.3%) in the neurological system. Gastrointestinal events
ranked second and constituted 26.7% of all events. The rest of the reported AEs do not exceed 10% each separately (general manifestations, 7.6%, respiratory system 6.5%, infection 6.3%, dermatological and pain, 5.6%, hematological, 4.2%). In conclusions, the treatment with, Nimotuzumab in combination with radiotherapy in pediatric patients with diffuse intrinsic brain stem astrocyte tumors, demonstrated an increase in the rate of progression-free survival (PFS) (72.9%) at 6 months treatment in relation to the historical rate (50%). The combination of radiotherapy and Nimotuzumab.

## P-99
THE PROGNOSTIC VALUE OF THE MELATONERGIC SYSTEM-BASED ASMT:CYP1B1 INDEX IN SOLID TUMORS

Kinker G.S.¹, Ostrowski L.H.¹, Oba-Shinjo S.M.², Muxel S.M.¹, Marie S.K.N.², Markus R.P.¹, Fernandes P.A.¹

¹Department of Physiology, Institute of Bioscience, University of São Paulo, São Paulo, Brazil; ²Department of Neurology, School of Medicine, University of São Paulo, São Paulo, Brazil

Melatonin synthesized and released by the pineal gland at night is responsible for translating the environmental dark phase to the organism. The production of melatonin has also been detected in many extra-pineal tissues, including retina, gastrointestinal tract and brain. Generally, extra-pineal melatonin is poorly released into the circulation, acting locally in an autocrine and paracrine manner. Currently, it is well established that melatonin exerts oncostatic effects across a wide variety of tumors, impairing tumor growth, invasion and angiogenesis. In this sense, we have recently demonstrated that the ability of gliomas to synthesize/accumulate melatonin negatively correlates with their overall malignancy (Kinker et al., 2016). Using the analysis of The Cancer Genome Atlas (TCGA) glioma RNAseq data, we designed a predictive model of the content of melatonin in the tumor microenvironment, the ASMT:CYP1B1 index, combining the gene expression levels of melatonin synthesis and metabolism enzymes. The ASMT:CYP1B1 index negatively correlates with glioma malignancy grade and is a grade- and histological type-independent prognostic factor. Additionally, the analysis of TCGA RNAseq data of 7,125 samples from 17 solid tumor types (including gliomas) demonstrated that the ASMT:CYP1B1 index negatively correlates with the expression of genes involved in the control of tumor growth, migration and invasion. Interestingly, many of these genes are known to have their expression modulated by melatonin, including COL1A1, ITGAV, ACTA2, IGF1, STAT3, HIF1A and SPHK1. More importantly, as observed for gliomas, a low ASMT:CYP1B1 index value, suggestive of reduced melatonin, is associated with poor survival in bladder urothelial carcinomas, cervical/endocervical cancers, colorectal adenocarcinomas, lung squamous cells carcinomas, pancreatic adenocarcinomas, paraganagliomas/pheochromocytomas, stomach adenocarcinomas, and endometrial cancer. Overall, our data reveal the prognostic value of the melatonergic system in solid tumors and support further investigations of the biological relevance of melatonin synthesized by malignant cells. Financial support: FAPESP (2010/52687-1, 2013/13691-1, 2014/27287-0), CNPq (480097/2013-5, 162670/2014-1).

**Keywords:** solid tumors, melatonin, ASMT, CYP1B1, molecular markers

## P-100
PHARMACOGENETIC STUDY FOCUSED ON FLUOXETINE PHARMACODYNAMICS IN CHILDREN AND ADOLESCENT PATIENTS: IMPACT OF THE SEROTONIN PATHWAY

Rodríguez N.¹, Mas S.¹,²,³, Blázquez A.⁴, Boloc D.¹, Lafuente A.¹,²,³, Arnaiz J.A.¹,²,⁵, Lázaro L.²,³,⁴,⁶, Gassó P.¹,²

¹Department of Basic Clinical Practice, University of Barcelona, Spain; ²Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; ³Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Madrid, Spain; ⁴Department of Child and Adolescent Psychiatry and Psychology, Institute of Neurosciences, Barcelona, Spain; ⁵Department of Clinical Pharmacology, Hospital Clinic de Barcelona, Barcelona, Spain; ⁶Department of Psychiatry and Clinical Psychobiology, University of Barcelona, Spain

**Background:** Although fluoxetine has been shown to be effective in children and adolescents, there is great inter-individual variability in clinical response. Pharmacogenetic studies of fluoxetine in pediatric patients are scarce. After reporting the effect of genetic variants in genes related to the fluoxetine pharmacokinetics on clinical response in a pediatric population, we now evaluate the impact of genetic markers involved in its pharmacodynamics.

**Methods:** Eighty-three children and adolescents aged between 10 and 17 years of age receiving fluoxetine treatment were recruited. Clinical improvement after 12 weeks of fluoxetine treatment was assessed by score changes in different scales including CDI, OCI-CV, SCARED, GAF, CGI-S and CGI-I. The genetic association analysis was performed with a total of 316 validated SNPs in 45 candidate genes involved in six different pathways directly or indirectly related to fluoxetine mechanism of action including the serotonergic and other neuromodulator systems, the hypothalamic-pituitary-adrenal axis, the circadian rhythm, and inflammatory and neurodevelopmental pathways.

**Results:** Clinical improvement after treatment with fluoxetine in our pediatric population was significantly associated with two polymorphisms located in genes related to the serotonergic system: the 5-hydroxytryptamine receptor 1B (HTR1B) and the tryptophan 5-hydroxylase 2 (TPH2). Particularly, clinical improvement was lower in heterozygous (AT) patients for rs130058 (HTR1B) and higher in minor allele homozygous (TT) for rs4570625 (TPH2).

**Conclusions:** Although a wide range of candidate genes related to different pathways were assessed, it seems that the most directly related pathway with the action mechanism of fluoxetine is the one that had the most important genetic effect on clinical response.

**Keywords:** children, fluoxetine, HTR1B, pharmacogenetics, polymorphism, TPH2

## P-101
PREVALENCE AND PATTERN OF POTENTIALLY INAPPROPRIATE MEDICATION IN OLDER PRIMARY CARE PATIENTS. PROMINENT ROLE OF BENZODIAZEPINES

Blanco-Reina E.¹, Rodríguez-Lavado I.¹, Aguilar-Cano L.², Valdellós J.³, Ariza-Zafra G.⁴, Ocaña-Riola R.⁵, García-Merino R.⁶, Bellido I.¹

¹Pharmacology Department, Málaga Biomedical Institute (IBIMA), University of Málaga, Spain; ²Health District of Málaga, Spain;

³Health District of Málaga Norte, Málaga, Spain; ⁴Geriatrics Department, University Hospital of Albacete, Albacete, Spain;

⁵Andalusian School of Public Health, Granada, Spain; ⁶Health District of Córdoba Sur, Córdoba, Spain

Potentially Inappropriate Medication (PIM) is one of the main risk factors for adverse drug events (ADEs) in older people. STOPP criteria (Screening Tool of Older Persons' Prescriptions) has proven greater correlations to ADEs with PIMs than with those defined by the Beers criteria. The aim of this work was to determine the prevalence and profile of PIMs and its predictive factors in elderly population.
**Methods:** This was a multicenter study with a cross-sectional design. The study population comprised 582 community-dwelling residents ≥65 years in Health District of Málaga. Data recorded included sociodemographic characteristics, clinical status, comorbidity, functional assessment and complete information about drugs intake (ATC classification, indication, dosage and length). The primary endpoint was the percentage of participants receiving at least one PIM according the recent update of STOPP criteria (v2, 2015).

**Results:** A total of 3626 prescriptions were analyzed. The mean age was 73.1 (±5.6) years (57.4% female). The median number of medications per patient was 6.8 drugs (range 0–23). 66.5% of the participants had prescribed at least one drug of N-group. The third part received benzodiazepines as usual treatment (long-acting: 15%). PIMs were detected in 66.8% of patients (51.7% if we exclude the most questionable criteria: lack of evidence-base clinical indication, drugs prescribed beyond the recommended duration and duplicate drugs). The most frequent PIMs were benzodiazepines for ≥4 weeks (31%) and drugs that predictably increase the risk of falls (benzodiazepines, neuroleptics, hypnotic Z-drugs and vasodilator drugs) (35%), followed by the use of opioids without concomitant laxative (8%), and long-term use of NSAID (>3 months) (2.7%).

**Conclusions:** The prevalence of PIM was high, and shows an increase over previous studies in Primary Care. Special attention should be paid on benzodiazepines, which keep being the most frequent PIM.

**Funding:** Consejería de Salud, Junta de Andalucía (Grant number: PI 0234/14)

**Keywords:** inappropriate prescribing; STOPP version 2; elderly adults; drug-related problems

## P-102

### EPIGENETIC AND GENETIC VARIANTS IN THE *HTR1B* GENE AND CLINICAL IMPROVEMENT IN CHILDREN AND ADOLESCENTS TREATED WITH FLUOXETINE

Gassó P.¹,², Rodríguez N.¹, Blázquez A.³, Monteagudo A.¹, Boloc D.¹, Plana M.T.³, Lafuente A.¹,²,⁴, Lázaro L.²,³,⁴,⁵, Arnaiz J.A.¹,²,⁶, Mas S.¹,²,⁴

¹Department of Pathological Anatomy, Pharmacology and Microbiology, University of Barcelona, Spain; ²Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; ³Department of Child and Adolescent Psychiatry and Psychology, Institute of Neurosciences. Hospital Clínic de Barcelona, Spain; ⁴Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Spain; ⁵Department of Psychiatry and Clinical Psychobiology, University of Barcelona, Spain; ⁶Clinical Pharmacology Department, Hospital Clínic de Barcelona, Spain

The serotonin 1B receptor (5-HT₁B) is important to both the pathogenesis of major depressive disorder and the antidepressant effects of selective serotonin reuptake inhibitors. Although fluoxetine has been shown to be effective and safe in children and adolescents, not all patients experience a proper clinical response, which has led to further study into the main factors involved in this inter-individual variability. Our aim was to study the effect of epigenetic and genetic factors that could affect 5-hydroxytryptamine receptor 1B (*HTR1B*) gene expression, and thereby response to fluoxetine. A total of 83 children and adolescents were clinically assessed 12 weeks after of initiating an antidepressant treatment with fluoxetine for the first time. We evaluated the influence of single nucleotide polymorphisms (SNPs) specifically located in transcription factor binding sites (TFBSs) on their clinical improvement. A combined genetic analysis considering the significant SNPs together with the functional variant rs130058 previously associated in our population was also performed. Moreover, we assessed, for the first time in the literature, whether methylation levels of the *HTR1B* promoter region could be associated with the pharmacological response. Two, rs9361233 and rs9361235, were significantly associated with clinical improvement after treatment with fluoxetine. The heterozygous genotype combination analysis showed a negative correlation with clinical improvement. The lowest improvement was

experienced by patients who were heterozygous for all three SNPs. Moreover, a negative correlation was found between clinical improvement and the average methylation level of the *HTR1B* promoter. These results give new evidence for the role of epigenetic and genetic factors which could modulate *HTR1B* expression in the pharmacological response to antidepressants.

**Keywords:** Fluoxetine, Pharmacogenetics, Methylation, Children, *HTR1B*, Polymorphism

## P-103

### HOST CHARACTERISTICS, PHARMACOLOGICAL PROPERTIES AND THEIR INTERACTIONS INFLUENCE TYPE OF LIVER INJURY IN HEPATOTOXICITY

Gonzalez-Jimenez A.¹, Chen M.², McEuen K.², Sanabria-Cabrera J.¹, Slim M.¹, Stephens C.¹, Suzuki A.³, Andrade R.J.¹, Lucena M.I.¹

¹IBIMA, H. Virgen de la Victoria, Universidad de Malaga, CIBERehd, Malaga, Spain; ²National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR; ³University of Arkansas for Medical Sciences, Little Rock, AR, United States

**Background:** The clinical course of drug-induced liver injury (DILI) is typically categorized as hepatocellular, cholestatic, or mixed. This categorization is important for the diagnosis of causative drugs and the assessment of possible clinical outcomes. Here we aimed to evaluate host and pharmacological factors that were associated with the development of liver injury pattern.

**Methods:** 564 DILI cases classified according to biochemical parameters as having hepatocellular and cholestatic type of liver injury were selected out of 701 cases enrolled in the Spanish DILI registry. Variate analyses and logistic regressions (R software) were performed to compare the pharmacological properties of causative drugs and host factors between the two groups (hepatocellular vs. cholestatic injury).

**Results:** Of the study cohort, 437 (77%) cases had hepatocellular and 127 (23%) cholestatic type of liver damage. Seven parameters were identified as being best predictors for liver injury pattern by using the logistic regression analysis. Parameters increasing the risk of hepatocellular damage included: age <60 years (Odds ratio or OR=2.06; *P* = 0.0029), previously known allergic records (OR=2.61; *P* = 0.0493), ≥50% hepatic metabolism (OR=1.70; *P* = 0.0417), enterohepatic circulation (OR=4.94; *P* < 0.0001) and causative drugs with a chemical double/single bond ratio >0.5 (OR=2.04; *P* = 0.0285). In contrast, presence of co-medications for cardiac disease and causative drugs with a higher number of heterorings (OR=0.68, *P* = 0.0270) were found to decrease the risk of a hepatocellular injury pattern (OR=0.47, *P* = 0.0030). Interaction between older age (≥ 60 y) and culprit drugs with low hepatic metabolism, but not high hepatic metabolism, increase the risk of cholestatic damage (OR=3.49, *P* = 0.0001).

**Conclusions:** Host factors and pharmacological properties seem to play a complementary role in the prediction of injury pattern in DILI. The pharmacological properties facilitating hepatocellular damage seem to be related with increased drug exposure or hepatocyte damage. A high drug metabolism could be associated with an increased possibility of generating reactive metabolites.

**Funding:** Fondos FEDER(PI15/01440); AEMPS; CIBERehd_by_ISCIII.

## P-104

### PREVALENCE OF UGT1A1 GENETIC VARIANTS IN ARGENTINEAN POPULATION, POTENTIAL IMPLICATIONS FOR PHARMACOGENOMIC TESTING

Scibona P.¹,², Belloso W.H.¹

¹Clinical Pharmacology Section, Internal Medicine Service; ²Instituto de Ciencias Básicas y Medicina Experimental (ICBME). Hospital Italiano de Buenos Aires. Buenos Aires, Argentina

**Introduction:** The uridine diphosphate glucuronosyltransferase (UGT) enzymes constitute a superfamily of enzymes responsible for the
glucuronidation of target substrates. The UGT family (UGT1A, UGT2, UGT3 and UGT8) is responsible for the glucuronidation of hundreds of compounds and drugs, which makes the enzymes in this group relevant to pharmacogenetic research. The UGT1A family is located on chromosome 2q37. There are genetics variants in the promoter region of *UGT1A1*, upstream to exon 1. The normal sequence of the TATAA element within the promoter is A[TA]6TAA. These variants generates longer version of the TATAA sequence, A[TA]7TAA (UGT1A1\*28), A[TA]8TAA (UGT1A1\*37), that cause reduced production of bilirubin-UGT. Since bilirubin-UGT is involved in the glucuronidation of several important drugs, individuals with UGT1A1\*28 variant may be more susceptible to the toxic effect of substances that require bilirubin-UGT-mediated hepatic glucuronidation prior to excretion. Is known this variant increase the risk of toxicity with irinotecan and the hyperbilirubinemia with atazanavir. Also there is a shorter version A[TA] 5TAA (UGT1A1\*36) that causes increased production of bilirubin-UGT.

**Objectives:** Determine the frequency of *UGT1A1* variants in Argentinean population.

**Methods:** 100 DNA samples from healthy volunteers were analyzed. The presence of variant UGT1A1\*28 was confirmed by direct sequencing of PCR products.

**Results:** Among the 100 subjects analyzed, 49% (49) were males and 51% (51) were females with a median age of 43 and a range of 32–80 years. The distribution of the UGT1A1 was: 70.5% (70.5) for the \*1 allele. for the 21.5% \*28 allele and \*36 allele 1%, 48% (48) presented the \*1/\*1 genotype, while 43% (43) had \*1/\*28, 2% (2) had \*1/\*36 and 7% (7) showed the \*28/\*28. No preferential sex distribution was observed in between genotypes.

Comment: The high prevalence of low-of-function alleles in argentinean general population provide support for further evaluation of the practical consequences of pharmacogenomic test.

## P-105

### DRUG INDUCED LIVER INJURY ASSESSMENT: ACUTE HEPATITIS DUE TO HEPATITIS E VIRUS, A DIAGNOSIS TO CONSIDER

*Sanabria-Cabrera J*¹, *Sanjuán-Jiménez R*¹, *Stephens C*¹, *Robles-Díaz M*¹, *Ortega-Alonso A*¹, *Jiménez-Pérez M*², *Medina-Cáliz I*¹, *Slim M*¹, *Andrade R.J*¹, *Lucena M.I*¹

¹UGC Aparato Digestivo y Servicio de Farmacología Clínica, Instituto de Investigación Biomédica de Málaga (IBIMA), Hospital Universitario Virgen de la Victoria, Universidad de Málaga, CIBERehd, SCReN, Málaga, Spain; ²UGC Aparato Digestivo, Instituto de Investigación Biomédica de Málaga (IBIMA), Hospital Universitario Regional de Málaga, Málaga, Spain

**Introduction:** Drug-induced liver injury (DILI) remains one of the most challenging diseases due to the absence of diagnostic tests and biomarkers. DILI usually presents as an acute hepatitis-like picture requiring extensive differential diagnosis, is a diagnosis of exclusion requiring prudent causality assessment. Hepatitis E virus (HEV), which is considered a rare condition in Spain, is not usually ruled out during acute hepatitis assessment.

**Material and Methods:** Analysis of a cohort of 180 patients from the Spanish DILI Registry diagnosed with DILI was undertaken. We analyzed HEV Immunoglobulin (Ig)G and M from two groups of serum samples based on the time point of collection (27 samples during the episode of liver damage and 153 at different time points after resolution). In patients showing anti-HEV-IgM+, AgHEV and RNA-HEV were performed.

**Results:** Out of 180 patients included, 60 (33, 3%) were tested positive for anti-HEV-IgG and 6 for anti-HEV IgM (1 positive for HEV-RNA and 2 for Ag-HEV). In the group of samples collected during the episode, 3/27 (11%) were positive for IgM-HEV.

<table><caption>Table 1. Demographic and clinical data of patients with positive IgM anti-HEV.</caption><thead><tr><th>Case<br>n°</th><th>Age<br>(years)</th><th>Sex</th><th>Drug</th><th>Latency<br>(days)</th><th>Peak<br>ALT(1)<br>or<br>AST(2)</th><th>HEV<br>RNA</th><th>Ag<br>HEV</th></tr></thead><tbody><tr><td>1</td><td>49</td><td>Female</td><td>paracetamol</td><td>9</td><td>1840 (2)</td><td>Negative</td><td>Positive</td></tr><tr><td>2</td><td>74</td><td>Male</td><td>cefditoren</td><td>40</td><td>4191 (1)</td><td>Positive</td><td>Positive</td></tr><tr><td>3</td><td>26</td><td>Female</td><td>derketoprofen</td><td>8</td><td>1561 (1)</td><td>Negative</td><td>Negative</td></tr><tr><td>4</td><td>56</td><td>Female</td><td>isoniazid</td><td>27</td><td>954 (1)</td><td>Negative</td><td>Negative</td></tr><tr><td>5</td><td>35</td><td>Male</td><td>erythromycin</td><td>27</td><td>2469 (1)</td><td>Negative</td><td>Negative</td></tr><tr><td>6</td><td>75</td><td>Male</td><td>amoxicillin</td><td>4</td><td>2967 (1)</td><td>Negative</td><td>Negative</td></tr></tbody></table>

**Conclusions:** Evidence of hepatitis E infection is present in a significant number of patients suspected to have DILI. Seroprevalence of IgG-HEV is also very high. HEV should be ruled out in all patients suspected to suffer from DILI in Spain.

Funding: ISCIII, FEDER, (PI15/01440, PI16/01748), Consejería de Salud (PI-0274-2016), AEMPS, CIBERehd, SCReN.

**Keywords:** hepatotoxicity; drugs; DILI; HEV

## P-106

### PROSPECTIVE DRUG-INDUCED LIVER INJURY REGISTRY: CLINICAL CHARACTERISTICS AND OUTCOMES OF 915 CASES

*Slim M*¹, *Sanabria J*¹, *Robles-Díaz M*¹\*, *Medina-Cáliz I*¹, *Sanjuán-Jiménez R*¹, *González-Jiménez A*¹, *Ortega A*¹, *García-Cortés M*¹\*, *García-Muñoz B*¹, *Fernandez M.C*², *Castiella A*³, *Zapata E*³, *Hallal H*⁴, *Conde I*⁵\*, *Prieto M*⁵\*, *Montane E*⁶, *María Morillas R*⁶, *Soriano G*⁷\*, *Roman E*⁷\*, *Navarro J.M*⁸, *Jimenez M*⁹, *Aldea A*¹⁰, *Hernández-Guerra M*¹⁰, *Gines P*¹¹, *Blanco S*¹², *Moreno J.M*¹³, *Romero-Gomez M*¹⁴\*, *Gomez-Dominguez E*¹⁵, *Andrade R*¹\*, *Lucena M.I*¹\*

¹Instituto de Investigación Biomédica de Málaga -IBIMA, Hospital Universitario Virgen de la Victoria, Universidad de Málaga, \*CIBERehd, Málaga, Málaga, Spain; ²Hospital Torrecardenas, Almería, Spain; ³Hospital de Mendaro, Guipuzkoa, Spain; ⁴Hospital Morales Meseguer, Murcia, Spain; ⁵Hospital Universitario y Politécnico La Fe. CIBERehd, Valencia, Spain; ⁶Hospital Germans Trias i Pujol, Badalona, Spain; ⁷Hospital de la Santa Creu i Sant Pau, CIBERehd, Barcelona, Spain; ⁸Agencia Sanitaria Costa del Sol, Málaga, Spain; ⁹Instituto de Investigación Biomédica de Málaga (IBIMA), Hospital Regional de Málaga, Málaga, Spain; ¹⁰Hospital Universitario de Canarias, Tenerife, Spain; ¹¹Hospital Clínic de Barcelona, CIBERehd, Barcelona, Spain; ¹²Hospital Universitario Basurto, Bilbao, Spain; ¹³Complejo Hospitalario de Albacete, Albacete, Spain; ¹⁴Hospitales Universitarios Virgen Macarena-Virgen del Rocío, CIBERehd, Sevilla, Spain; ¹⁵Hospital 12 de Octubre, Madrid, Spain

**Introduction:** Idiosyncratic drug induced liver injury (DILI) is a rare adverse drug reaction that poses major challenges to healthcare practitioners and regulatory agencies. We aimed to update the clinical characteristics and outcomes of DILI patients, and the drugs frequently implicated in hepatotoxicity in Spain.

**Methods:** We analyzed 915 DILI cases (842 single-episodes, 55 re-challenges, 18 double-episodes) in 857 patients included in the Spanish Registry from 1994 to 2015. Cases were compared according to pattern of liver damage (hepatocellular, HC; cholestatic, Chol or mixed, Mix).

**Results:** Median age was 57 years with mean body mass index of 25.8 ± 3.8 kg/m2. HC, Chol and Mix patterns of liver damage were identified in 65%, 18% and 17% of cases, respectively. More than half of the cases were of moderate severity (58%). Patients with Chol and Mix pattern were older (median 64 y and 62 y, respectively) than HC patients (median 52 y), *P* < 0.001. Median time to DILI onset was 30 days in the HC group compared to 20 and 17 days for Chol and Mix, respectively, *P* < 0.001. The median time to resolution was
111 days, which was similar among the different patterns of liver injury. Jaundice and hospitalization were more common in Chol cases compared to HC (77% and 70% vs. 65% and 54%, *P* < 0.05, respectively). HC cases exhibited greater risk of developing fatal/severe outcomes, 15% vs. 7% and 3% in Chol and Mix cases, respectively, *P* < 0.001. Anti-infectives, central nervous system, cardiovascular and anti-inflammatory agents were the most commonly implicated therapeutic classes accounting for 37%, 14%, 11% and 9% of cases, respectively.

**Conclusions:** The pioneering prospective Spanish DILI Registry proved to be valuable for in-depth phenotyping of hepatotoxicity. It also constitutes an important tool for public health promotion in post-marketing drug surveillance.

**Funding:** Research grants from AEMPS and FEDER (PI15/01440, PI16/01748). CIBERehd by ISCIII.

### Pharmacogenomic Alerts in an Electronic Medical History

Simonovich Ventura; Rebrij Romina; Scibona Paula; Belloso Waldo; Luna Daniel; Otero Carlos; Gonzalez Bernaldo de Quiros Fernan

### Hospital Italiano de Buenos Aires

The human genome has different variants in each individual. This variability implies different responses to the same drug. The doctor who indicates a treatment may lack the specific knowledge about the effects that a drug can produce against the genetic variants of the patient. For this reason it is considered necessary the existence of a system of pharmacogenomic alerts as a tool to support the physician when making a prescription through an electronic medical record. The present work describes the development of the necessary components for the functioning of this system: a genetic database of patients, a pharmacogenomic knowledge base, an inference engine and a user interface. These components integrate a system that considers the genetic variants of the patient and verifies if there is any risk before the prescription of a drug, issuing an alert to the doctor if necessary. A system of pharmacogenomic alerts in an Electronic Clinical History has the potential to reduce adverse effects in people highly susceptible to suffering them.

P-107

### OLEUROPEIN EXHIBITED ANTI-INFLAMMATORY AND ANTI-OXIDANT ACTIVITIES IN IL-1β-STIMULATED HUMAN SYNOVIAL FIBROBLASTS

Castejón M.L., Rosillo M.Á., Montoya T., González-Benjumea A., Fernández-Bolaños J.M., Alarcón-de-la-Lastra C.

Departamento de Farmacología, Facultad de Farmacia, Universidad de Sevilla

Rheumatoid arthritis (RA) is a chronic and systemic inflammatory autoimmune disease mainly characterized by aggressive hyperproliferation of synovial fibroblasts (SFs) and massive infiltration of inflammatory immune cells inducing a progressive matrix degradation, destruction of cartilage and bone erosion through the production of inflammatory mediators. Oleuropein, is the most prevalent phenolic component in olive leaves, seed, pulp and peel of unripe olives and is responsible for unprocessed olives characteristic bitter taste. This secoiridoid possesses a well-documented pharmacological properties, including antioxidant and anti-inflammatory, consequently is available as food supplement in Mediterranean countries. However, at the date, anti-arthritic effects of oleuropein on SFs have not been yet elucidated. Thus, the aim of the present study was to investigate the potential effects of oleuropein, on IL-1β-induced inhibitory production of inflammatory mediators and oxidative stress in human synovial sarcoma cell line (SW982). In order to gain a better insight into mechanisms of action, signaling pathways were also explored.

Cell viability was determined using sulforhodamine B (SRB) assay. The expression of inflammatory cytokines IL-6, TNF-α, MMP-1 and MMP-3 was evaluated by ELISA. Moreover, changes in the protein expression of cyclooxygenase (COX)-2, microsomal prostaglandin E synthase-1 (mPGES-1) as well as mitogen-activated protein kinase (MAPKs), nuclear factor kappa B (NF-κB), and nuclear factor

e2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) signalling pathways were analysed by western blot.

Oleuropein exerted anti-inflammatory and anti-oxidant effects via down-regulation of MAPKs and NF-κB signaling pathways and induction of Nrf2-linked HO-1 controlling the production of inflammatory mediators decreasing IL-6 and TNF-α cytokines, MMP-1 and MMP-3 levels as well as mPGES-1 and COX-2 overexpression. Thus, oleuropein might provide a basis for developing a new dietary strategy for the prevention and management of RA.

P-108

### IKKα IS INVOLVED IN PRO-INFLAMMATORY ACTIVITY OF EXTRACELLULAR NICOTINAMIDE PHOSPHORIBOSYLTRANSFERASE (ENAMPT) IN BONE MARROW-DERIVED MACROPHAGES

Montserrat-de la Paz S.¹,²,³, Martín Rubio M.E.⁴, Navarro P.D.¹,²,³,⁴, Biessen E.A.L.³, Bermudez B.³,⁴

¹Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa, CSIC, Spain; ²Department of Pharmacology, School of Pharmacy, University of Seville, Spain; ³Department of Pathology, Maastricht University Medical Center, Maastricht University, The Netherlands;

⁴Department of Cell Biology, Faculty of Biology, University of Seville, Spain

**Introduction:** The IKKα, a subunit of the NF-κB-activating IKK complex, has emerged as an important regulator of inflammatory gene expression. Although intracellular nicotinamide phosphoribosyltransferase (iNAMPT) is a key enzyme in controlling NAD⁺ metabolism, circulating eNAMPT has been associated with several metabolic and inflammatory disorders, including cancer and cardiovascular diseases. Herein, the potential role of IKKα and the underlying mechanisms by which eNAMPT could exert the metabolic and inflammatory dysfunctions were investigated.

**Material and Methods:** Wild type (C57BL/6) and IKKα⁻/⁻ (C57BL/6 IKKαAA/AA) mice were used in this study. Bone marrow (BM) was isolated from wild type and IKKα⁻/⁻ bones, then cells were pooled and cultured in RPMI supplemented with 15% L929-conditioned medium to generate BM-derived macrophages (BMDMs) and ex vivo studies were performed. These BMDMs were pretreated with FK866 (an iNAMPT inhibitor), Lentivirus iNAMPT (LV.iNAMPT), and eNAMPT treatment before the isolation of RNA and cell supernatants. mRNA levels of genes related to IKKα pathway, macrophage polarization, inflammation and secreted pro-inflammatory cytokines were determined by RT-qPCR and ELISA respectively.

**Results:** IKKα⁻/⁻ BMDMs pretreated with eNAMPT, in contrast to those from wild type, revealed a significant decrease in pro-inflammatory (Tnfa, Il1b, and Il6) gene expression and cytokine (TNF-α, IL-1β, and IL-6) release. Moreover, wild type BMDMs pretreated with eNAMPT were polarised towards M1 phenotype, whereas IKKα⁻/⁻ BMDMs were skewed to an unaltered macrophage phenotype compared to IKKα⁻/⁻ BMDMs in absence of eNAMPT. Finally, pre-incubation of BMDMs with LV.iNAMPT enhanced the pharmacological benefits of IKKα inhibition; increasing the expression of PPARγ-related genes.

**Conclusion:** These findings provide evidence that NF-κB play a role in pro-inflammatory activity of eNAMPT and reveal that targeting IKKα kinase activity represents a pharmacological approach.

P-109

### THE ANGIOTENSIN-(1-7)/ MAS RECEPTOR AXIS COUNTERACT PRO-INFLAMMATORY SIGNALING IN HUMAN VASCULAR SMOOTH MUSCLE CELLS

Romero A., Villalobos L., San Hipólito Á., Ramos M., Cercas E., Vallejo S., Romacho T., Carraro R., Sánchez C., Peiró C.

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

**Background and Aims:** Vascular inflammation is the main characteristic of vascular diseases such as hypertension and atherosclerosis,
which are one leading causes of death worldwide. For this reason, tar-
geting inflammation is nowadays regarded as a challenging pharmaco-
logical strategy to prevent or delay the development of vascular
diseases. Angiotensin (Ang)-(1-7) is a heptapeptide of the renin-angio-
tensin system (RAS) that binds Mas receptors and participates in vas-
cular homeostasis by antagonizing some of the actions of Ang II.
Here, we explored the capacity of Ang-(1-7) to counteract human aor-
tic smooth muscle cell (HASMC) inflammation triggered by both (Ang
II) or RAS-independent stimuli, such as interleukin (IL)-1β.

**Methods:** HASMC cultures were isolated from aortic fragments obtained from organ donors at Hospital Universitario de Getafe. The expression of inducible nitric oxide (iNOS) and the release of nitric oxide were determined by Western blot and the Griess method, respectively. The activation of NADPH oxidase was determined by luciferin-derived chemiluminescence, while NF-κB activation was measured by electromobility shift assay.

**Results:** Ang-(1-7) inhibited in a concentration-dependent manner the iNOS induction and NO release elicited by Ang II and IL-1β. The effect of Ang-(1-7) was equally blocked by two different Mas receptor antagonists, A77 and D-Pro-Ang-(1-7), suggesting the participation of a unique Mas receptor subtype. Both Ang II and IL-1β were able to activate NADPH oxidase and nuclear factor (NF)-κB upstream of iNOS induction, and such pro-inflammatory signaling was attenuated by Ang-(1-7).

**Conclusion:** Ang-(1-7) can act as a counter-regulator of the inflamma-
tion of vascular smooth muscle cells triggered by Ang II, but also by
other stimuli beyond the RAS. Activating the Ang-(1-7)/Mas axis may
represent a pharmacological opportunity to attenuate the pro-inflamma-
tory environment that promotes and sustains the development of vascu-
lar diseases.

P-110
EVOO PHENOLS, HYDROXYTYROSOL AND HYDROXYTYROSOL ACETATE, SUPPRESSED IL-1β-INDUCED PROINFLAMMATORY MEDIATORS PRODUCTION IN HUMAN SYNOVIAL FIBROBLASTS

Angeles R.M., Marina S.-H., Luisa C.M., Tatiana M., Alejandro G.-B., María F.-B.J., Catalina A.-de-la.-L.

Department of Pharmacology, Faculty of Pharmacy, University of Sevilla

The hallmark of rheumatoid arthritis (RA) pathology is characterized
by both hyperproliferation of synovial fibroblasts (SFs) and massive
infiltration of inflammatory immune cells, including CD4 + T-cells
and innate immune cells. It is well known that various proinflamma-
tory mediators, including IL-6, IL-1β, TNF-α, and MMPs, released
from RA-SFs are involved in the destruction of both articular bone
and cartilage. This study was conducted to evaluate the efficacy of
hydroxytyrosol (HTy) and hydroxytyrosol acetate (HTy-Ac), phenolic
compounds from EVOO in regulating IL-1β-induced production of
metalloproteases (MMP-1 and MMP-3), IL-6 and TNF-α in human
synovial cell line, SW982. Treatment with HTy and HTy-Ac signifi-
cantly inhibited IL-1β -induced MMPs, IL-6 and TNF-α production
when measured by ELISA. The nuclear transcription NF-κB represent
an attractive target for RA since it induces the transcription of
inflammatory cytokines and mediators (IL-6 and MMPs) being
responsible in addition to phosphorylation of MAPKs and COX-2
up-regulation. The effects of HTy and HTy-Ac on COX-2 and m-
PGEs1 protein expression and the activation of MAPKs and NF-κB
were also examined in SW982 cells by western blotting IL-1β-up
regulation COX-2 and m-PGEs1 were diminished by HTy and HTy-
Ac. IL-1β-induced p38, JNK and ERK1/2 phosphorylation were
inhibited by HTy and HTy-Ac treatment. Similarly, the phenolic
compounds inhibited IL-1β-induced NF-κB activation. These results
suggest that these EVOO phenols reduce the production of proinflam-
matory mediators in SW982 cells; the mechanism underlying these
protective effects could be related to the inhibition of MAPKs and
NF-κB signalling pathways.

P-111
VASCULAR DAMAGE INDUCED BY VISFATIN IN MICE
INVOLVES NADPH OXIDASE ACTIVATION AND
PROSTANOID RELEASE

Ramos-Gonzalez M.¹, Vallejo S.¹, San Hipólito-Luengo A.¹, Romero A.¹, Cercas M.¹, Valencia I.¹, Gómez-Cerezo J.², Peiró C.¹, Sánchez-Ferrer C.F.¹

¹Departamento de Farmacología, Universidad Autónoma de Madrid,
Facultad de Medicina, Madrid, Spain; ²Departamento de Medicina
Interna, Hospital Infanta Sofía, Madrid, Spain

**Introduction:** Vascular aging, obesity, and type 2 diabetes mellitus are associated with high circulating levels of pro-inflammatory adipocytokines, namely IL (interleukin)-1β or visfatin. We investigated whether the *in vivo* infusion of these adipocytokines can produce vascular alterations mimicking those observed in aging or metabolic diseases.

**Methodology:** Osmotic mini-pumps were implanted in three-month-
old male mice, and vehicle, visfatin (100 ng/kg/day) alone or with the
specific inhibitor of visfatin enzymatic activity FK866 (2.4 mg/kg/
day), or IL-1β (12 µg/kg/day) were infused for 7 days. Some mice
also received i.p. the IL-1β-receptor antagonist anakinra (100 mg/kg/
day) for 3 days. Reactivity of mesenteric arteries was studied *ex vivo*
using a small vessel myograph. Microvessels from visfatin-treated mice
were preincubated *ex vivo* with 10 µM apocynin (NADPH oxidase
inhibitor), 10 µM SQ-29,548 (thromboxane A2 receptor antagonist) or
100 µg/ml anakinra. Moreover, microvessels from control mice were
treated *ex vivo* with visfatin (50 ng/ml) alone or in the presence of the
previously described inhibitors.

**Results:** The infusion of visfatin or IL-1β reduced the endothelium-
dependent relaxations produced by acetylcholine (ACh, 10 nM-
10 µM). The endothelial dysfunction induced by visfatin was reversed
by the simultaneous *in vivo* infusion of FK866 or by the *ex vivo* pre-
incubation of the segments with apocynin, SQ-29,548, or anakinra,
indicating a role for superoxide anions, vasoconstrictor prostanoids,
and endogenous pro-inflammatory IL-1β, respectively. Anakinra infu-
sion also prevented IL-1β-induced endothelial dysfunction. Visfatin
evoked endothelial dysfunction when acutely exposed *ex vivo* to
mesenteric fragments from control mice, and this effect was blocked
by apocynin and SQ-29,548, but not with anakinra. None of the tested
drugs modified the contractility to 3 µM noradrenaline or the endothe-
lium-independent vasodilations induced by sodium nitroprusside
(10 nM-10 mM).

**Conclusion:** The infusion of the adipocytokines visfatin or IL-1β pro-
duces endothelial dysfunction in mice, norporting their possible role as
mediators of vascular damage and premature aging associated with
metabolic diseases.

**Keywords:** adipocytokines, vascular damage, visfatin

P-112
PROSTANOID-MEDIATED INHIBITION OF IL-6 TRANS-
SIGNALLING IN PULMONARY ARTERIAL HYPERTENSION:
A ROLE FOR EPAC-1 MEDIATED INDUCTION OF
'SUPPRESSOR OF CYTOKINE SIGNALLING 3' (SOCS3)

Durham G., Nasim T., Palmer T.

School of Pharmacy, University of Bradford, Bradford, UK

Pulmonary arterial hypertension (PAH) is a devastating disease of the pulmonary vasculature associated with endothelial cell (EC) dysfunction, vascular inflammation and remodelling, which contribute to eventual heart failure and death. Recent research has highlighted inflammation as a key factor in PAH development, in particular the cytokine interleukin-6 (IL-6). IL-6 signals via a JAK/STAT signalling pathway to induce transcription of a number of pro-inflammatory and pro-angiogenic genes that enable PAH to progress, as well as the transcription of suppressor of cytokine signalling 3 (SOCS3) which is involved in a negative feedback loop that limits IL-6 signalling.
Current PAH therapies include prostanoid drugs which act by stimulat-
ing intracellular 3',5'-cyclic adenosine monophosphate (cAMP) levels.
cAMP has been well documented as an inhibitor of endothelial dys-
function via exchange factor directly activated by cAMP (EPAC)1
mediated induction of SOCS3.

My studies are testing the hypothesis that an important mechanism by
which cAMP-mobilising prostanoid drugs limit PAH is by inhibiting
IL-6-mediated pulmonary inflammation and remodelling via Epac1-
mediated SOCS3 inhibition of IL-6 induced JAK/STAT signalling. We
have demonstrated that prostanoid drugs beraprost and treprostinil both
induce SOCS3 mRNA and protein in pulmonary arterial ECs to inhibit
IL-6 mediated Tyr705 phosphorylation of STAT3 by 30% ±8
(P < 0.01) and 25% ±9 (P < 0.05) respectively for n = 4 experiments.
We will also present data assessing the functional significance of pros-
tanoid mediated inhibition of IL-6 signalling in pulmonary arterial
ECs, and determine the EPAC1 and SOCS3 dependence of their inhi-
bitory effects on IL-6 signalling.

In summary, from these and future studies, it is anticipated that more
effective strategies will emerge with which to target the IL-6/JAK/
STAT signalling pathway in PAH.

**Keywords:** Pulmonary Arterial Hypertension; Cytokine Signalling; Jak-STAT; cAMP

P-113

HYDROXYTYROSOL DERIVATIVE MODULATES
INFLAMMATORY RESPONSE IN LPS-INDUCED MURINE
PERITONEAL MACROPHAGES

Montoya T.¹, Aparicio-Soto M.¹, Sánchez-Hidalgo M.¹, Begines P.²,
Fernández-Bolaños J.G.², Alarcón-de-la-Lastra C.¹

¹Department of Pharmacology, Faculty of Pharmacy, University of Seville, Seville, Spain; ²Department of Organic Chemistry, Faculty of Chemistry, University of Seville, Seville, Spain

**Background:** The beneficial effects of extra virgin olive oil have been corroborated by a large number of epidemiologic studies. Among the different components of olive oil, its phenolic compounds such as hydroxytyrosol (HTy), seem to have a special role in these beneficial effects. The present study was designed to investigate specially the antioxidant and anti-inflammatory role of HTy and its derivate in lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages.

**Methods:** Isolated murine peritoneal macrophages were treated with HTy or its derivative in the presence or absence of LPS (5 µg/ml) for 18 h. Cell viability was determined using sulforhodamine B (SRB) assay and nitric oxide (NO) production was measured using the Griess reaction. Supernatants were collected, and then it were used to determined cytokines production, such as IL-1β, TNF-α, IFN-γ, IL-17 and IL-6, using Enzyme-linked immunosorbent assay (ELISA) kits. Pro-inflammatory enzymes and transcription factors expression were detected by Western blotting.

**Results:** HTy and its derivative reduced significantly nitrites levels and induced a significant decrease on inducible nitric oxide synthase (iNOS) expression. Moreover, HTy-derivative showed better results in the reduction of the most of the studied cytokines when compared with HTy. However, only HTy down-regulated significantly cyclooxygenase (COX)-2 protein expression. Our findings demonstrated no significant changes in p38 and JNK protein phosphorylation after treatments, suggesting that the MAPKs signalling pathway does not make a consistent contribution in the anti-inflammatory activities of HTy-derivative. However, the treatment with HTy and its derivative produced a significant up-regulation of Nrf-2 and HO-1 expression compared with LPS-DMSO cells. In the same way, both of them showed a significant reduction of STAT-3 expression.

**Conclusions:** This study establishes that HTy and its derivative could improve LPS-induced oxidative stress and inflammatory response reducing NO generation by down-regulation of iNOS and COX-2 protein expression via inhibition of Nrf-2 and HO-1 signaling pathway.

P-114
ANTI-INFLAMMATORY AND IMMUNOMODULATORY
EFFECTS OF OSTEOSTATIN IN THE CIA MODEL

Nacher-Juan J., Terencio M.C., Alcaraz M.J., Ferrándiz M.L.

Pharmacology Department and IDM, University of Valencia, Valencia, Spain

Parathyroid hormone related protein (PTHrP) and its derived peptides have been recently studied for the treatment of bone diseases. One of them, osteostatin (PTHrP(107-111)), has shown *in vitro* anti-inflammatory and anti-senescence properties (1). The aim of the present study was to examine the effects of osteostatin in an animal model of rheumatoid arthritis.

We used the collagen-induced arthritis (CIA) model in DBA1/J mice. After the onset of arthritis, we administered 40 µg/kg/day and 80 µg/kg/day s.c. of osteostatin for 13 days. On day 14, serum was obtained to determine cytokines and bone metabolism mediators by Multiplex assay. After the sacrifice of mice, lymph nodes were extracted to characterize lymphocyte population by flow cytometry and cell proliferation by BrdU assay. Limbs were collected and homogenized to determine cytokines by ELISA.

Results demonstrated a dose-related improvement of the arthritic macroscopic score after 13 days of treatment with osteostatin. IL-1β (11731 ± 2535 pg/ml) and IL-17 (81.91 ± 22.34 pg/ml) levels in arthritic control paw homogenates were significantly decreased by osteostatin at 80 µg/kg/day (5319 ± 1016 pg/ml, *P* < 0.05 and 45.24 ± 5.88 pg/ml, *P* < 0.05, respectively). IL-6 levels in serum of control mice (2653 ± 601 pg/ml) were also reduced at the same dose (1482 ± 127 pg/ml, *P* < 0.05). In addition, osteostatin treatment significantly reduced lymphocyte proliferation (≥50% inhibition) with respect to the cells obtained from control arthritic lymph nodes and the decrease of CD4⁺/CD8⁺ ratio observed in control mice was reverted in treated groups. These results confirm the ability of osteostatin to reduce inflammatory and immune responses in an *in vivo* model of chronic inflammation and suggest its potential interest for rheumatoid arthritis treatment.

1. Platas J, Guillén MI, Gomar F, Castejón MA, Esbrit P, Alcaraz MJ. Anti-senescence and Anti-inflammatory Effects of the C-terminal Moiety of PTHrP Peptides in OA Osteoblasts. J Gerontol A Biol Sci Med Sci. 2016;0(0):1-8.

**KeyWords:** arthritis, osteostatin, *in vivo*, CIA
Funded by SAF2013-48724R (MINECO, FEDER) and PROMETEOII/
2014/071 (GENERALITAT VALENCIANA).

P-115
PSORIATIC FIBROBLASTS POLARIZE MACROPHAGES TO
A PRO-INFLAMMATORY PHENOTYPE

Arasa J.¹, Andrés R.M.¹, Valcuende-Cavero F.², Payá M.¹, Terencio M.C.¹, Montesinos M.C.¹

¹Department of Pharmacology and IDM, University of Valencia, Valencia, Spain; ²Department of Dermatology, University Hospital La Plana, Vila-real, and Predepartamental Unit of Medicine, Universitat Jaume I, Castellón, Spain

Fibroblasts are mesenchymal cells that regulate tissue homeostasis and are involved in the switch from acute resolving to chronic inflammation (1). We have recently described that psoriatic dermal fibroblasts showed a defective activation of the JNK pathway that resulted in diminished cytokine production and STAT3 signaling (2). In the present study we aimed to determine if fibroblast-released factors influence the polarization of macrophages.

Healthy and psoriatic fibroblasts were incubated during 24 h with IL1-
β and supernatants were collected to obtain the correspondent healthy
(HF) or psoriatic (PF) conditioned media, which were added to macro-
phage-derived THP-1 cells. After 24 h, levels of TNF-α and IL-10
were determined by ELISA. Macrophages incubated with PF condi-
tioned media produced high levels of TNF-α (369.5 ± 27.5 pg/ml vs.
51.5 ± 17.5 pg/ml in untreated cells *P* < 0.001 *n* = 5–8) and low levels of IL-10 (63.4 ± 3.1 pg/ml *P* < 0.0001 *n* = 5–10). In contrast, macrophages incubated with HF conditioned media, produced lower levels of TNF-α (161.0 ± 21.8 pg/ml) and higher levels of IL-10 (213.0 ± 11.4 pg/ml). Psoriatic fibroblast showed a defect in COX-2 induction, which resulted in reduced PGE₂ production. Consistently, conditioned media of HF previously treated with indomethacin significantly increased TNF-α (574.9 ± 74.7 pg/ml *P* < 0.0001 *n* = 5–8) and reduced IL-10 (45.8 ± 5.3 pg/ml *P* < 0.0001 *n* = 5–10) production by THP-1. These results were corroborated directly stimulating macrophages with IL-1β in the presence or absence of exogenous PGE₂.

Our results suggest that healthy fibroblasts support the M2 anti-inflammatory phenotype in macrophages, which is lost in psoriatic fibroblasts. Therefore, dermal fibroblast could play a relevant role in the perpetuation of psoriasis by favouring the pro-inflammatory activation of macrophages while hindering resolution of inflammation. Additionally, our findings offer a possible explanation of the disease exacerbation triggered by NSAIDs.

(1) Buckley CD (2016). Rheumatology (Oxford). https://doi.org/10.1093/rheumatology/kew289.

(2) Arasa J et al. (2015). *Exp Dermatol.* 24:800–2.

**Keywords:** psoriasis, macrophages, fibroblasts, COX-2

P-116

BENEFICIAL EFFECT OF ADENOSINE A₂B RECEPTOR ACTIVATION IN A MURINE MODEL OF SKIN HYPERPLASIA

Marín A., Nacher-Juan J., Catalán L., Payá M., Montesinos M.C., Terencio M.C.

Pharmacology Department and IDM, University of Valencia, Valencia, Spain

It is known that adenosine mediates, at least in part, the anti-psoriatic effect of methotrexate via interaction with one or more of four known cell-surface receptors (A₁, A₂A, A₂B and A₃). Recently, we have demonstrated that adenosine regulates inflammatory and proliferative response in human keratinocytes through A₂B receptor activation (1). In the present study we determine the effect of topical application of BAY60-6583 (BAY), a selective A₂B agonist, on the inflammatory murine model of epidermal hyperplasia induced by the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA).

BAY (1–10 µg/site), or vehicle (acetone) were applied on the shaved backs of female Swiss mice 30 minutes before TPA (2 nmol/site) for three consecutive days. The next day, animals were sacrificed and 1 cm² punch biopsies were collected, weighed and either homogenized or processed for histological analysis.

Topical application of BAY (10 µg/site) produced a significant reduction of score and edema determined by the weight of biopsies (115.7 ± 4.6 mg vs. 147.3 ± 9.3 mg in vehicle-treated mice, *n* = 8–10, *P* < 0.01). Treatment with the A₂B receptor agonist also reduced the levels of PGE₂ (4.6 ± 1.8 ng/ml vs. 10.6 ± 2.9 ng/ml in vehicle-treated, *n* = 5–8, *P* < 0.05) in skin homogenates. The possible effect on inflammatory skin infiltrate was observed in H&E stained paraffin-embedded tissue sections and correlated with the reduction of myeloperoxidase activity determined in homogenates (A₄₅₀ nm 0.568 ± 0.025 vs. 0.374 ± 0.025 in vehicle-treated *n* = 5–8 *P* < 0.05).

These results suggest that activation of the adenosine A₂B receptor, besides reducing keratinocyte proliferation, could regulate the inflammatory response in skin effect and therefore constitute a possible therapeutic strategy in psoriasis.

(1) Andrés RM *et al*, (2017). Adenosine A₂A and A₂B Receptors Differentially Modulate Keratinocyte Proliferation: Possible Deregulation in Psoriatic Epidermis. *J Invest Dermatol*.137,123-131.

**Keywords:** adenosine, A₂B receptor, mouse skin, BAY60-6583

P-117

SUPPRESSOR OF CYTOKINE SIGNALLING 3 (SOCS3) INTERACTION WITH CAVIN-1 LINKS SOCS3 FUNCTION AND CAVIN-1 STABILITY

Williams J.J.L.¹, Alotaiq N.², Mullen W.², Burchmore R.³, Liu L.⁴, Baillie G.S.², Schaper F.⁵, Pilch P.F.⁴, Palmer T.M.¹

¹School of Pharmacy, University of Bradford, Bradford, UK; ²Institute of Cardiovascular and Medical Sciences; ³Polyomics Facility, University of Glasgow, Glasgow, UK; ⁴Departments of Biochemistry and Medicine, Boston University School of Medicine, Boston, USA;

⁵Department of Systems Biology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany

Caveolae are lipid raft microdomains essential for the compartmentalisation and regulation of several signalling pathways e.g. JAK/STAT signalling. Disruption of caveolae is a significant factor in multiple disorders including muscular dystrophy, cardiovascular disease, and cancer. Central to caveolae stability is cavin-1 which couples caveolae to the microtubule network to prevent degradation of a key structural element i.e. caveolin-1, and caveolae disassembly. Via an unbiased quantitative proteomics screen, we have identified SOCS3, a negative regulator of JAK/STAT signalling, as a novel cavin-1 interactor.

SOCS3-cavin-1 interactions were characterised by immunoprecipitation assays and probing overlapping peptide arrays. SOCS3 bound to multiple regions within cavin-1, while a PEST motif within the C-terminal region of the SOCS3 SH2 domain was required for interaction with cavin-1 independently of its capacity to bind phospho-tyrosine. Biochemical analysis and confocal imaging also demonstrated that SOCS3 localisation within lipid raft microdomains and at the plasma membrane required cavin-1. Interestingly, SOCS3 does not ubiquitinate cavin-1 but instead supports cavin-1/caveolae stability. Moreover, genetic deletion of cavin-1 results in the loss of SOCS3-mediated inhibition of cytokine signalling. Importantly, while the inhibitory function of SOCS3 relies on its induction, caveolae stabilisation occurs at basal SOCS3 expression levels. Thus, transmission electron microscopy demonstrated that SOCS3 knock-out endothelial cells show reduced levels of caveolae.

Our data suggest a novel role for SOCS3 in regulating caveolae assembly while cavin-1, acting as a scaffold-protein, might aid SOCS3-dependent regulation of JAK/STAT signalling. This is the first indication of a novel role for SOCS3 in caveola homeostasis and suggests that loss of caveolae represents a novel mechanism by which chronic activation of pro-inflammatory JAK/STAT signalling could be triggered in disease.

**Keywords:** JAK/STAT, caveolae, cavin-1, SOCS3, inflammation

P-118

CX₃CL1/CX₃CR1 AND CCL2/CCR2 AXES ARE POTENTIAL THERAPEUTIC TARGETS TO PREVENT ARTERIAL LEUKOCYTE ADHESION IN METABOLIC SYNDROME

Marques P.¹,², Collado A.¹, Martinez-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; ³University Clinic Hospital-CIBERDEM, Department of Medicine, University of Valencia, Valencia, Spain

**Introduction and Objectives:** Metabolic syndrome (MS) is characterized by a cluster of physiological alterations that collectively convey in a low grade of systemic inflammation and endothelial dysfunction. Both are involved in atherosclerotic lesion formation yet the pathways involved in its development remain unknown. CX₃CL1/CX₃CR1 and CCL2/CCR2 axes have emerged as potential candidates for cardiovascular disease (CVD) prediction and targets for therapeutic intervention. Therefore, the potential link of these chemokine/receptor axes and MS-induced endothelial dysfunction were investigated.
**Material/Methods:** Whole blood samples from 20 MS patients and 20 age-matched controls were analysed by parallel-plate flow chamber assay, to evaluate platelet-leukocyte and leukocyte adhesion to TNF-α-stimulated arterial endothelium. Flow cytometry was employed to determine platelet (P-selectin expression and % of PAC-1⁺ platelets), monocyte (CD11b expression) and lymphocyte (CD69 expression) activation. CX₃CR1 expression was also determined on platelets, monocyte subtypes (classical or M1, intermediate/pro-inflammatory or M2 and non-classical or M3) and lymphocyte subsets (CD4⁺ and CD8⁺) by flow cytometry.

**Results:** Enhanced platelet-leukocyte and leukocyte adhesiveness to TNFα-stimulated arterial endothelial cells was found in MS patients vs. age-matched controls. Neutralization of CX₃CL1 or CCL2 activity resulted in significant inhibition of adhesive interactions to the dysfunctional arterial endothelium in MS patients but not in control volunteers. In MS patients, greater platelet activation and CX₃CR1 expression than age-matched controls was detected. Additionally, MS patients presented augmented numbers of platelet-CD8⁺ lymphocyte aggregates, enhanced lymphocyte activation and increased CX₃CR1 expression than control subjects. Monocyte activation and the percentage of CX₃CR1 expressing platelet/M1 monocyte aggregates were also increased in MS patients. These observations correlated with the augmented platelet-leukocyte- and leukocyte-arterial adhesiveness in MS.

**Conclusions:** Given that platelet/leukocyte- and leukocyte-arterial adhesion precedes atherogenic process development, blockade of either CX₃CL1/CX₃CR1 or CCL2/CCR2 axis may ameliorate the risk of suffering further cardiovascular events in patients with this metabolic disorder.

**Keywords:** Metabolic syndrome; Cardiovascular; Inflammation, chemokine/receptor axes

P-119

CHONDROITIN SULFATE INHIBITS MONOCYTE CHEMOATTRACTANT PROTEIN-1 RELEASE FROM 3T3-L1 ADIPOCYTES: A NEW TREATMENT OPPORTUNITY FOR OBESITY-RELATED METABOLIC SYNDROMES?

*Montell E.<fnref n="1" />, Stabler T.V.², Vergés J.<fnref n="1" />, Kraus V.B.²,³*

**Background:** Monocyte chemoattractant protein-1 (MCP-1) overproduction from inflamed adipose tissue is a major contributor to obesity-related metabolic syndromes. We have recently published that chondroitin sulphate (CS) can attenuate the monosodium urate (MSU) crystal mediated THP-1 macrophage inflammatory response through an inhibitory effect on NF-κB activation.

**Objective:** We sought to determine whether CS had a similar inhibitory effect on MCP-1 release from lipopolysaccharide (LPS) stimulated adipocytes.

**Methods:** We treated 3T3-L1 adipocytes with LPS together with CS (Bioibérica, Spain) in a physiologically relevant range (10–200 µg/ml) and 24 h after we measured MCP-1 release (R&D Systems, Minneapolis, MN, USA). We also cultured THP-1 monocytes and tested whether CS (200 µg/ml) could inhibit cell migration induced by 24 h exposure to human recombinant MCP-1 (R&D Systems; 0, 3.125–100 ng/ml).

**Results:** We found that LPS (1 µg/ml) caused a significant rise in MCP-1 release (*P* < 0.0001) from 3T3-L1 adipocytes. CS in physiologically achievable concentrations (100–200 µg/ml) produced a dose dependent reduction (*P* < 0.01 at 100 µg/ml and *P* < 0.001 at 200 µg/ml) of MCP-1 release from 3T3-L1 adipocytes in response to LPS. Recombinant MCP-1 (25–100 ng/ml) caused a dose dependent increase (*P* < 0.001 at 25 ng/ml and *P* < 0.0001 at 100 ng/ml) in cell migration of THP-1 monocytes. CS at the highest test concentration (200 µg/ml) had no effect on MCP-1 mediated THP-1 migration.

**Conclusions:** Our data demonstrate that CS inhibits the release of MCP-1 from 3T3-L1 adipocytes stimulated with LPS, but has no effect on the chemotactic action of MCP-1 on THP-1 monocytes. Furthermore, our work data strongly suggests that it is the inhibition of MCP-1 release by CS that underlies this effect and not a direct inhibition of the chemotactic action of MCP-1 by CS. Given the importance of MCP-1 over-production in obesity-related metabolic syndromes, the recruitment blocking of macrophages to adipose tissue exerted by CS, could provide a new treatment opportunity for these syndromes.

**Keywords:** metabolic syndromes; obesity; adipocytes; chondroitin sulfate; monocytes, macrophages, inflammation

P-120

A FLUOROMETHYLCHALCONE DERIVATIVE AS INHIBITOR OF CASPASE-1 ACTIVATION IN CPPD-INDUCED MOUSE AIR POUCH MODEL

Catalán L., Terencio M.C., Ferrándiz M.L., Alcaraz M.J.

Pharmacology Department and IDM, University of Valencia, Valencia, Spain

The inflammatory process includes the participation of many factors, such as the pro-inflammatory cytokines IL-1β and IL-18. Both require post-translational processing by active caspase-1 to generate their active forms. Caspase-1 activity is controlled by inflammasomes, a group of cytosolic protein complexes whose activity has been linked to many diseases including arthritis. This complex is activated by microbial pathogens and damage-associated molecular patterns such as calcium pyrophosphate dihydrate crystals (CPPD)<fnref n="1" />.

Previous studies demonstrated the potential anti-inflammatory profile of 3,4,6-trimethoxy-6'-trifluoromethylchalcone (CH) by inhibition of NO release and iNOS expression in macrophages². In the present study, we have characterized the involvement of caspase-1 activation in the CPPD-induced mouse air pouch model (MAP) in order to determine the anti-inflammatory effect of CH.

Six days after the initial air injection, CH (100 µg/pouch) was injected in the treated group. After 20 minutes, CPPD (100 mg/pouch) was injected in CH and control group. Mice were sacrificed 6 h later and pouch exudates were collected. Cells were measured using a Coulter Counter. IL-1β, IL-18 and TNF-α were determined by ELISA in supernatants. Cell pellet was used to measure the active p20 subunit of caspase-1 by Western Blotting.

Results demonstrated that CH treatment down-regulated cell migration into exudates (218.00 ± 29.90 cells/ml *P* < 0.05) with respect to control group (502.10 ± 75.32 cells/ml). IL-1β (517.10 ± 59.20 pg/ml) and IL-18 (94.94 ± 12.75 pg/ml) levels in control supernatants were also significantly decreased after CH treatment (IL-1β: 279.40 ± 32.68 pg/ml *P* < 0.01); IL-18: 51.39 ± 1.64 pg/ml *P* < 0.05). Besides, the increase in the expression of activated p20 subunit from control exudates demonstrated the involvement of caspase-1 in MAP, suggesting that inhibition of inflammasome activation is as a potential mechanism of CH anti-inflammatory effect.

1. Martinon *et al.* (2006), *Nature*, **440**:237–41.

2. Rojas *et al.* (2002), *Bioorg Med Chem Lett*, **12**:1951–4.

**Keywords:** Inflammation; Caspase-1; CPPD; Air Pouch Model
Funded by SAF2013-48724R (MINECO, FEDER) and PROMETEOII/2014/071 (GENERALITAT VALENCIANA).

<footnote n="1">¹Pre-Clinical R&D Area, Bioibérica S.A.U., Barcelona, Spain; ²Duke Molecular Physiology Institute, Duke University School of Medicine, Durham, USA; ³Division of Rheumatology, Duke University School of Medicine, Durham, USA</footnote>
P-121
VLDL AND APOLIPOPROTEIN CIII INDUCE ER STRESS AND INFLAMMATION AND ATTENUATE INSULIN SIGNALING VIA TOLL-LIKE RECEPTOR 2 AND ERK1/2 IN SKELETAL MUSCLE CELLS

Vázquez-Carrera M., Botteri G., Montori M., Gumà A., Pizarro J., Barroso E., Cedó L., Escolà-Gil J.C.

University of Barcelona; University of Barcelona; University of Barcelona; University of Barcelona; University of Barcelona;
University of Barcelona; Hospital de Sant Pau, Barcelona; Hospital de Sant Pau, Barcelona

**Aim/hypothesis:** Here, we examined whether very low-density lipoproteins (VLDL) and apolipoprotein (apo)CIII induce endoplasmic reticulum (ER) stress, inflammation, and insulin resistance in skeletal muscle.

**Methods:** Studies were conducted on mouse C2C12 myotubes, isolated skeletal muscle, and in skeletal muscle from transgenic mice overexpressing apoCIII.

**Results:** C2C12 myotubes exposed to VLDL showed increased levels of ER stress and inflammatory markers, whereas peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) and AMP-activated protein kinase (AMPK) levels were reduced and the insulin signaling pathway was attenuated. VLDL effects were also observed in isolated skeletal muscle incubated with VLDL. The changes caused by VLDL were dependent on extracellular signal-regulated kinase (ERK1/2) because they were prevented by the ERK1/2 inhibitor U0126 or by knockdown of this kinase by siRNA transfection. ApoCIII mimicked the effects of VLDL and its effects were also blocked by ERK1/2 inhibition, suggesting that this apolipoprotein was responsible for the effects of VLDL. Skeletal muscle from transgenic mice overexpressing apoCIII showed increased levels of some ER stress and inflammatory markers and of phosphorylated ERK1/2 levels, whereas PGC-1α levels were reduced, confirming apoCIII effects in vivo. Finally, incubation of myotubes with a neutralizing antibody against toll-like receptor 2 abolished the effects of apoCIII on ER stress, inflammation, and insulin resistance, indicating that the effects of apoCIII were mediated by this receptor.

**Conclusions/interpretation:** These findings indicate that VLDL and apoCIII induce ER stress, inflammation, and insulin resistance by activating ERK1/2 through TLR2, suggesting that targeting VLDL overproduction can ameliorate insulin resistance and it might prevent type 2 diabetes mellitus.

P-122
ORM2 AND APOA2 SERUM LEVELS CAN PREDICT OA PATIENT RESPONSE TO CHONDROITIN SULFATE/GLUCOSAMINE HYDROCHLORIDE: RESULTS FROM THE MOVES STUDY

Calamia V.¹, Camacho M.¹, Gonzalez L.¹, Fernández-Puente P.¹, Rego-Pérez L.¹, Herrero M.², Martinez H.², Ruiz-Romero C.¹, Blanco F.J.¹,³

¹Grupo de Proteomica-PBR2-ProteoRed/ISCIII-Servicio de Reumatología, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Universidade da Coruña, A Coruña; ²Clinical R&D Area, Bioiberica S.A., Barcelona, Spain; ³Unidad de Investigación Clínica-Servicio de Reumatología, Complexo Hospitalario Universitario de A Coruña (CHUAC), A Coruña

**Background:** A shotgun proteomic analysis performed on sera from patients enrolled in the Multicentre Osteoarthritis INTERVENTion trial with Sysadoa(MOVES) led to the discovery of a panel of predictive protein biomarkers useful to stratify osteoarthritis(OA) patients into responders and non-responders, either to Chondroitin sulfate/

glucosamine hydrochloride(Droglican®, Bioiberica S.A., Barcelona, Spain) or Celecoxib.

**Objectives:** To validate the sensitivity and specificity of a panel of six serum proteins useful to predict the patient response to Droglican treatment.

**Methods:** We analyzed the serum levels of: APOA2, APOA4, APOH, C4BPa, ITIH1 and ORM2 by enzyme-linked immunosorbent assays (ELISAs). All the subjects studied belonged to the MOVES cohort at baseline (Droglican sub-cohort, *n* = 260). Non-parametric and multivariate analysis were performed to test the effects of the clinical variables gender, age, BMI, radiologic Kellgren/Lawrence(K/L) grade and WOMAC score at baseline, as well as the biomarker serum levels, on the response to Droglican treatment according to the OMERACT-OARSI criteria and the WOMAC pain score (20%, 30%, 50% and 70% reduction) recorded at the end of the trial (after 6 months of treatment).

**Results:** We found decreased serum levels of ORM2 at baseline in responders to Droglican according to the OMERACT-OARSI criteria vs. non-responders (76,11 ± 53,25 vs 104,25 ± 84,93; *n* = 171 vs 46; *P* = 0.047). APOA2 appeared statistically increased in responders with a 50% reduction in WOMAC pain score compared to non-responders (79,95 ± 58,53 vs 66,05 ± 46,49; *n* = 129 vs 112; *P* = 0.028). Patients with lower levels of ORM2 (median concentration= 69.8 µg/ml) and higher level of APOA2 (median concentration= 63.8 µg/ml) responded better to pharmacotherapy. Statistical interactions between ORM2 and APOA2 levels and radiologic K/L grade were also detected (*P* = 0.048 and *P* = 0.002, respectively). No statistically significant differences were found for the other four proteins.

**Conclusion:** Our results show that ORM2 and APOA2 levels significantly correlates with patients response to Droglican suggesting the possibility of their use in predictive assays in order to optimize therapeutic outcomes in OA. Validation studies in different cohorts are needed to identify and validate a cut-off point for these biomarkers.

P-123
EVALUATION OF THE ANTI-INFLAMMATORY ACTIVITY OF LXR AGONISTS IN *IN VITRO* AND *IN VIVO* MODELS

López R.¹, Morales O.¹, Pagán I.¹, Montero J.L.¹, Andrés M.², Taltavull J.², Godessart N.¹, Balagué C.¹

¹Skin Biology and Pharmacology; ²Medicinal Chemistry & Screening, Almirall R&D, Sant Feliu de Llobregat, Spain

Evidence in the literature supports a role for topical liver X receptor (LXR) agonists in reducing skin inflammation and enhancing skin barrier function in different mouse models, making LXR an interesting target for treating skin inflammatory diseases such as atopic dermatitis. However, most evidence has been generated using non-selective LXR agonists. In this work we have assessed the effect of selective LXR agonists in several *in vitro* and *in vivo* mouse models of skin inflammation. Inhibition of cytokine secretion in human keratinocytes (HaCaT cell line and primary keratinocytes) and immune cells (PBMCs and THP-1 cell line) in response to a pro-inflammatory stimulus (TNF-α and Poly(I:C) + IL-4 for keratinocytes and LPS for immune cells) was analyzed. Gene expression analysis of different markers of skin inflammation and barrier function was performed in a commercial *in vitro* model of atopic dermatitis using skin equivalents stimulated with a mixture of cytokines (IL-4, IL-13, IL-22, TNF-α). Efficacy was evaluated through the topical route in mouse models of TPA-induced contact dermatitis and oxazolone-induced delayed type hypersensitivity (DTH), the latter considered a more relevant model of atopic dermatitis. We observed a poor anti-inflammatory activity *in vitro* and a lack of therapeutic effect in the DTH model. Only a dose-dependent effect was observed in the TPA-induced acute inflammation model. In addition, LXR agonists did not modify biomarkers of skin barrier function in human skin equivalents. These results call into question the potential of LXR agonists as a therapeutic option for treating skin inflammatory diseases such as atopic dermatitis.

**Keywords:** LXR; atopic dermatitis; inflammation; skin barrier function
P-124

CHARACTERIZATION OF THE IN VITRO AND IN VIVO PROFILE OF EPIGALLOCATECHIN-3-GALLATE

Carreño C.¹, Montero J.L.¹, Blanco A.¹, Jover I.², Espinosa S.²,
Kirsch A.³, Strahinjic I.³, Herbig M.³, Gavaldà A.¹, Godessart N.¹,
Balagué C.¹

¹Skin Biology and Pharmacology; ²Medicinal Chemistry and
Screening, Almirall, R&D Centre, St. Feliu de Llobregat, Spain;

³Pharmaceutical Development, Almirall Hermal GmbH, R&D Centre,
Reinbek, Alemania

Epigallocatechin-3-gallate (EGCG) is the main polyphenol in green tea reported to have anti-inflammatory, anti-carcinogenic, antioxidant and antimicrobial activities. A potential use for EGCG in different skin conditions has been put forward. The aim of this study was to characterize the pharmacological activity of EGCG in *in vitro* and *in vivo* models of skin inflammation. The anti-inflammatory effect of EGCG was assessed in human primary keratinocytes by the ability to inhibit the production of TSLP induced by Poly:IC/IL4, and in mice by the ability to reduce oxazolone-induced ear edema in a DTH-type reaction. In addition, cutaneous local tolerance was assessed in mice upon topical repeated administration for 4 days. In both *in vivo* models, EGCG formulated as an ointment (3%) or solubilized in ethanol (1, 3 and 10%) was compared to the commercial green tea extract approved for warts treatment (Veregen®) containing around 5% EGCG). EGCG modestly inhibited TSLP production in human primary keratinocytes with an IC₅₀ of 1.7 µM. However, assessment of the stability of EGCG demonstrated that it was completely degraded in the assay medium, suggesting that EGCG may not be responsible for this activity. In addition, no anti-inflammatory effect of two stable formulations (ointment and ethanol-based) was observed in the oxazolone-induced DTH model, whereas Veregen significantly increased ear edema. Local tolerance studies did not reveal any relevant clinical, macroscopic or histological changes compared to vehicle treated animals in the 4-day scheme. Taken together, these results question the therapeutic use of EGCG for inflammatory dermatoses and warrant further discussion.

**Keywords:** Epigallocatechin-3-gallate (EGCG); Veregen®; keratinocyte; skin inflammation models

P-125

DISCOVERY OF SMALL MOLECULES FOR IL-17
MEDIATED INFLAMMATORY DISEASES

Álvarez-Coiradas E., Gómez-Touriño I., Loza M., Domínguez E.

BioFarma Group, CiMUS Biomedical Research Institute, University of Santiago de Compostela, Santiago de Compostela, Spain

Inflammation is an adaptive response of the body to harmful stimuli. Chronic inflammation is the classic sign of autoimmune diseases and is associated with the malfunction of tissue. Such diseases have a great impact in the quality of life and the outcomes of patients, since they occur at early age. Although treatments have improved significantly with the introduction of biologics, novel small molecule approaches may enable further development of therapeutics.

Lately, interest has been sparked in interleukin-17 (IL-17), which binds to surface receptors with dimer structure to activate inflammation, as a novel pharmacological target for inflammatory autoimmune diseases. Therapeutic antibodies selectively targeting IL-17A have been approved for clinical use in psoriasis, a condition characterized by abnormal accumulation of skin cells (psoriatic plaques) due to overactivation of the immune system. Recently, a class of macrocyclic compounds was discovered, indicating that the IL-17 druggability with molecules might be feasible.

We aim to identify chemical tools to study the IL-17 mediated inflam-
matory mechanisms in psoriasis. For this purpose, we developed a pro-
tocol for the production of recombinant IL-17A in mammalian cells
that can be used in label-free binding assays. IL-17A was cloned into

pcDNA3.1.+ vector containing a C-terminal His₆ tag. The plasmid was transfected in HEK 293 cells and supernatants, where IL-17A is released, were collected after 48 hours. Subsequently, protein was purified by affinity chromatography on Ni-Sepharose Fast Flow column. We confirmed IL-17A expression by means of western blotting and we achieved a protein production of > 85% purity.

In order to identify novel ligands for IL-17A, we used DMR (Dynamic Mass Redistribution) technology. We tested the protein immobilization at different concentrations and pH, obtaining suboptimal binding levels. Further assay development and screening may provide a discovery platform for novel IL-17A ligands.

**Keywords:** inflammation; psoriasis; IL-17; label-free

P-126

EFFECT OF THE INTAKE OF THE STRAIN
BIFIDOBACTERIUM ANIMALIS SUBSP. LACTIS CECT 8145
IN ZÜCKER FATTY RATS

Amaya A.M., Noemí L.-C., Patricia M., Empar C., Salvador G., Daniel R.

Departamento de Farmacología, Facultad de Medicina, Universidad Complutense de Madrid, Avenida Complutense s/n, Madrid, Spain;
Departamento de Farmacología, Facultad de Medicina, Universidad Complutense de Madrid, Avenida Complutense s/n, Madrid, Spain;
Department of Food Biotechnology

We evaluate the effect of oral administration of the strain Bifidobacterium animalis subsp. lactis CECT 8145 in Zücker fatty rats. The Zücker fatty rats were randomly divided into two groups of 10 animals that were respectively fed with either B. animalis subsp. lactis CECT 8145 (10¹⁰ CFU/day) suspended in skim milk, or skim milk (control group), from week 5 until week 17 of life. A lean Zücker rat group (standard group) was included to provide normal values for the Zücker strain. To this group was administered skim milk for the same experimental period as Zücker fatty rats. Body weight gain was more accentuated in the fatty control group than in the fatty rats that received daily B. animalis subsp. lactis CECT 8145. In line, dry and liquid food intakes significantly decreased in the Zücker fatty group fed with this bacterial strain. These rats also showed decreased plasma ghrelin levels as compared with the Zücker fatty control group. Bifidobacterium animalis subsp. lactis CECT 8145 intake also decreased the pro-inflammatory cytokine tumoral necrosis factor-alpha and the oxidative stress biomarker malondialdehyde. Moreover, the ratio plasma total cholesterol/plasma cholesterol transported by high-density lipoproteins, considered as an indicative index for cardiovascular disease, also significantly decreased in the Zücker fatty rats that were fed with B. animalis subsp. lactis CECT 8145. On the contrary, this bacterial strain significantly increased plasma adiponectin (an insulin-sensitizing adipokine) and slightly improved plasma triglycerides and glucose metabolism biomarkers. Further research would be needed to support the effects of B. animalis subsp. lactis CECT 8145 in humans as a probiotic for metabolic syndrome prevention. Nevertheless, our results are promising and set the health and anti-obesity properties of this bacterial strain.

Supported by Biópolis S.L. (78/2012 U.C.M Project, art. 83 L.O.U.).
P-127
EFFECTS OF PEPSIN EGG WHITE HYDROLYSATE ON METABOLIC SYNDROME

Miguel M., Moreno-Fernández S., García-Rimón M., Cristina G., Esperanza H., Aleixandre M.A., Antonio U.J., Visitación L.-M., Gema V.

Instituto de Investigación en Ciencias de la Alimentación (CIAL, CSIC-UAM), Madrid, Spain Ciencias Básicas de la Salud, Facultad de Ciencias de la Salud, Universidad Rey Juan Carlos, Alcorcón, Madrid, Spain Facultad de Medicina, Universidad Complutense de Madrid

Metabolic syndrome (MetS) is a complex disorder which refers to the clustering of central obesity, insulin resistance, impaired glucose tolerance, hypertension and dyslipidemia. The prevalence of this disorder is increasing fast, and it is becoming an important and worldwide public health problem. The purpose of this study was to evaluate the effect of the administration of a pepsin egg white hydrolysate (HEW) on some metabolic complications developed in MetS. Thirty-four 8-week old Wistar rats where used and divided in four groups: Control group (C); Control + HEW group (CH); high-fat diet with a 25% dextrose solution (MS) and high-fat diet with a 25% dextrose and HEW 1 g/kg/day (MSH). Body weight, food and liquid intake were estimated weekly. The occurrence of neuropathic sign (tactile allodynia) was assessed once every 6 weeks using the von Frey hair test. At the end of the study, the abdominal circumference was registered. Blood samples for biochemical determinations (plasma glucose, lipid metabolism and oxidative stress biomarkers), and epididymal adipose tissue and liver for histopathological analyses were collected. Abdominal circumference, body, adipose tissue and liver weights were decreased after HEW consumption in MSH group. Plasma glucose values were slightly reduced in MS animals after HEW intake, and the tactile allodynia was significantly improved in this experimental group. The oxidative stress biomarkers, such as plasma malondialdehyde and liver glutathione were also decreased in MSH group, and both groups fed with HEW (CH and MSH) showed lower adiponectin levels. No significant differences were observed in the antioxidant capacity and lepin levels in plasma. Our results suggest that HEW may improve simultaneously several metabolic complications developed in MetS, and could be used as an alternative therapy to control the MetS progress in obese patients.

Supported by MINECO (AGL212-32387).

P-128
ANTIOXIDANT AND BIOACTIVE PROPERTIES OF JASONIA GLUTINOSA (COMPOSITAE) IN CAENORHABDITIS ELEGANS

Moliner C.<fnref n="1" />, Les F.<fnref n="1" />, Reigada I.<fnref n="1" />, Valero M.S.<fnref n="2" />, Langa E.<fnref n="1" />, Gómez-Rincón C.<fnref n="1" />, López V.<fnref n="1" />

Jasonia glutinosa (Compositae) is an endemism from the Iberian Peninsula traditionally used as a medicinal herbal tea to improve digestion and gastrointestinal disorders. The plant is known as té de roca (rock tea) or té de Aragón and has been highly cited in ethnopharmacological studies as a medicinal plant. Previous studies from our group have demonstrated the spasmolytic activity. We have now measured the antioxidant and protective effects of Jasonia glutinosa extract using *in vitro* and *in vivo* procedures as the living organism *Caenorhabditis elegans*. The antioxidant activity in living organisms was evaluated using *C. elegans* pre-treated with the plant extract and exposed to juglone as oxidizing agent. Life span of *C. elegans* treated with increasing concentrations of the plant extract was also tested. Antioxidant activity against free radicals was performed using enzymatic systems such as xanthine oxidase or monoamine oxidase as well as the free radical DPPH. Jasonia glutinosa

exerted antioxidant and protective properties both in cell free systems (enzymatic and chemical assays) and in *C. elegans* stressed with juglone. *Jasonia glutinosa* was able to scavenge free radicals in a dose dependent manner and to significantly increase *C. elegans* survival up to 10% compared to control groups, where all individuals died after 24 h of juglone treatment. Life span of *C. elegans* significantly augmented when worms were treated with 100 µg/ml of extract. We can conclude that the increase in life span and in the survival of *C. elegans* exposed to juglone could be mediated through antioxidant mechanisms of compounds present in *Jasonia glutinosa* extract.

**Acknowledgements:** Universidad San Jorge is thanked for financial support.

P-129
DUAL EFFECTS OF RESVERATROL ON CELL DEATH AND PROLIFERATION OF COLON CANCER CELLS

San Hipólito-Luengo A.<fnref n="1" />, Alcaide A.<fnref n="2" />, Ramos-González M.<fnref n="1" />, Cercas E.<fnref n="1" />, Vallejo S.<fnref n="1" />, Romero A.<fnref n="1" />, Talero E.<fnref n="2" />, Sánchez-Ferrer C.F.<fnref n="1" />, Motilva V.<fnref n="2" />, Peiró C.<fnref n="1" />

Colorectal cancer remains a main cause of death worldwide, and novel agents have been searched as polyphenols which have emerged as promising therapeutic tools in cancer. Resveratrol (3,5,4'-trihydroxy-trans-stilbene) induces cell death in different tumor cell lines, but it also stimulates the proliferation of specific breast and prostate cancer cell lines. Here, we studied the impact of resveratrol over a 100-fold concentration range on cell death and proliferation of HT-29 colorectal adenocarcinoma cells. A biphasic pattern was observed after 96 h of exposure to resveratrol. At lower concentrations (1 and 10 µmol/L), resveratrol promoted an increase in cell number. However, enhancing the resveratrol concentration up to 50 or 100 µmol/L resulted in reduced cell number, together with increased percentage of apoptotic or necrotic cells, thus indicating cytotoxicity. These proliferative properties at lower concentrations were shared by the flavonoid polyphenol quercetin. However, on a second colon cancer cell line, such as HCT116, the growth promoting properties of resveratrol could not be observed. The cytotoxic effect of resveratrol on HT-29 cells was associated with the over-activation of the superoxide-generating enzyme NADPH oxidase and increased levels of histone γH2AX, a marker of DNA damage. A parallel enhancement in the levels of sirtuin 6 was also observed, probably as a repair mechanism to counterbalance the damaged exerted by resveratrol to the cells. In conclusion, resveratrol seems to be an effective tool to be used in anti-tumor chemotherapy. However, the biphasic behavior of resveratrol indicates that this polyphenol may in some conditions favor tumor cell growth. Thus, appropriate local concentrations must be achieved to minimize unwanted harmful effects of resveratrol as an anti-cancer drug.

P-130
SAFETY STUDY ON THE USE OF MEDICINAL PLANTS

Mabel Rosalía G., Alicia Lilian T., Margarita B., Ana Elena G.

Universidad Nacional del Chaco Austral, Argentina

**Summary:** Since ancient times, man has used medicinal plants for the prevention and treatment of various diseases. Despite their beneficial and traditional role in different communities, it must be taken into consideration that they can also cause adverse reactions and toxicity. The aim of this study was to analyze the reports on Communication of Adverse Events for the use of Herbal Medicines, Plant Products and/or Preparations of Vegetable Drugs, received as Chaco's Peripheral Reporter for the National Pharmacovigilance System, part of the National Administration of Drugs, Food and Medical Technology (ANMAT) of Argentina during the 2010-2016 period. A retrospective

<footnote n="1">¹Department of Pharmacy, Universidad San Jorge, Villanueva de Gállego (Zaragoza), Spain; ²Departamento de Farmacología y Fisiología, Facultad de Veterinaria, Universidad de Zaragoza, Zaragoza, Spain</footnote>

<footnote n="1">¹Department of Pharmacology, Universidad Autónoma de Madrid;</footnote>

<footnote n="2">²Department of Pharmacology, Universidad de Sevilla</footnote>
analysis of the reports received from pharmacies and health centers was made using the Anatomical, Therapeutic, Chemical (ATC) classification guide for herbs. The causality was assessed by the Naranjo et al. algorithm and the severity was classified into three categories: mild, moderate, and severe. From the total of notifications received (n = 135), 80% involved the use of a single plant product, 15% the mixture of two or more and 5% the use of herbal medicines. Thirty-four percent of reports about adverse reactions involved children, while female sex prevailed over male. According to the ATC classification of herbs, 71% corresponded to the HA group (alimentary tract and metabolism) as the most reported, followed by 14% to the HN group (nervous system) and the rest to other groups. The most common adverse reactions proved to be probables and milds. The most frequently reported medicinal plants involving children were *Chenopodium ambrosioides* L. and *Illicium verum* Hook. F., with severe adverse effects such as seizures being described, whereas in the adult population it was *Cassia angustifolia* Vahl., with moderate adverse effects. It is essential to monitor and identify the risks posed by plants, especially when used by vulnerable populations, thereby promoting their safe use.

P-131
DIOSMETINE AND DIOSMINE INHIBIT THE
PROINFLAMMATORY CYTOKINES RELEASE

Zaragozá C.¹, Mantecón C.¹, Monserrat J.², Villaescusa L.¹, Álvarez-Mon M.², Zaragozá F.¹

¹Biomedical Sciences Department, Alcalá University, Madrid, Spain;

²Medicine Department, Alcalá University, Madrid, Spain

Nowadays cardiovascular diseases are major causes of death in the developed world. Platelets play a key role in thrombus formation. Experiments carried out in our laboratory showed diosmetine and diosmine, polyphenols of flavonoids family, had a potential antiplatelet activity. Pro-inflammatory cytokines promote the activity of specific adhesion molecules on the vascular endothelium contributing to the leukocyte rolling and their extravasation from capillaries, what leads to exacerbate the thrombosis clinical course. Therefore, we tested the impact of these products on the inhibition of these cytokines release.

**Material/Methods:** Whole blood aliquots extracted from 10 healthy volunteers were incubated with diosmetine/diosmine, both to 0.5 mM, 1 mM, and 2 mM concentration. Lipopolysaccharide (LPS) was added for inducing pro-inflammatory cytokines release. Samples were centrifuged and supernatant was collected in order to measure the IL-1β and TNF-α secretion from monocytes using specific immunoassay techniques.

**Results:** LPS (10 mg/ml) induced a significant increase on the IL-1β and the TNF-α release compared to control values. This production

was inhibited by diosmetine and diosmine at 0.5 mM, 1, and 2 mM concentrations. IL-1β inhibition mean with diosmetine was 21.94%± 3.38, 43.04%± 4.91, 71.05%± 3.44, and with diosmine was 0.00%, 0.00%, 1.51%±1.99 respectively. TNF-α inhibition mean with diosmetine was 43.36%± 1.89, 77.00%± 2.06, 84.11%± 2.12, and with diosmine was 0.00%, 7.91%± 2.61, 15.60%± 2.37, respectively.

**Conclusions:** Diosmetine was able to inhibit the pro-inflammatory cytokines released induced by LPS in a dose-dependent way, while diosmine provokes only certain effect over TNF-α at its higher dose. The diosmine antiplatelet activity was higher than diosmetine. The activity differences shown could be related with the different chemical structures of the heteroside and the aglycon. However these studies suggest both flavonoids could have a potential therapeutic use in cardiovascular disease.

**Keywords:** diosmetine, diosmine, IL-1β, TNF-α

P-132
ANTHELMINTIC ACTIVITY OF ESSENTIAL OILS AGAINST
ANISAKIS SIMPLEX L3 LARVAE

Cascella M.¹,², Gómez-Rincón C.¹, Langa E.¹, Maggi F.², López V.¹

¹Department of Pharmacy, Universidad San Jorge, Villanueva de Gállego (Zaragoza), Spain; ²School of Pharmacy, University of Camerino, Camerino, Italy

Anisakis simplex is a foodborne pathogen with increasing interest due to the high consumption of raw fish products and the prevalence of anisakiasis worldwide. Nineteen essential oils obtained from medicinal and culinary plants were screened against *Anisakis simplex* L3 larvae isolated from blue whiting fish (*Micromesistius poutassou*). The experiments were performed in small Petri dishes containing 10 larvae. The percentage of anisakis mortality after 48 h of treatment at 1 µl/ml was superior to 50% only for *Cinnamomum zeylanicum*, *Cinamomum cassia*, *Tanacetum annum* and *Origanum compactum*. Inhibition of the enzyme acetylcholinesterase through a colorimetric assay in 96-well plates was used to elucidate the pharmacological mechanism. The four essential oils were able to inhibit acetylcholinesterase, which could explain in part the mechanism of action against the parasites. The median lethal doses (DL₅₀) after 24 and 48 h were lower than 0.5 µl/ml for *C. zeylanicum*, *C. cassia* and *O. compactum*, revealing a good potential as anthelmintic natural products.

**Acknowledgements:** Pranarom International is thanked for financial support and supplying essential oils.

<table><caption>Table 1.</caption><thead><tr><th rowspan="3"></th><th colspan="4">Baseline</th><th colspan="4">6 months</th></tr><tr><th colspan="2">No bDNA</th><th colspan="2">bDNA</th><th colspan="2">No bDNA</th><th colspan="2">bDNA</th></tr><tr><th>CS/GH</th><th>CE</th><th>CS/GH</th><th>CE</th><th>CS/GH</th><th>CE</th><th>CS/GH</th><th>CE</th></tr></thead><tbody><tr><td>Adrenaline (pg/ml)</td><td>98 (88-127)</td><td>100 (77-118)</td><td>92 (78-114)</td><td>98 (75-115)</td><td>78* (65-92)</td><td>81* (62-92)</td><td>76* (58-97)</td><td>77* (66-105)</td></tr><tr><td>Noradrenaline (pg/ml)</td><td>1357 (790-1693)</td><td>1228 (738-1967)</td><td>1723# (1385-2381)</td><td>2765# (1232-3250)</td><td>1050 (811-1600)</td><td>1588 (882-2133)</td><td>1563 (1209-2180)</td><td>1528 (1174-2229)</td></tr><tr><td>IL-8 (pg/ml)</td><td>5.5 (1.3-9.4)</td><td>5.6 (1.7-14)</td><td>4.8 (3.9-5.7)</td><td>11 (1.9-15)</td><td>3.3* (0.0-11)</td><td>0.0* (0.0-5.9)</td><td>6.1 (3.7-14)</td><td>5.6 (1.5-16)</td></tr><tr><td>VAS score (0-100)</td><td>72 (66-82)</td><td>75 (65-81)</td><td>79 (67-90)</td><td>76 (70-80)</td><td>32* (22-45)</td><td>20* (10-60)</td><td>37* (27-50)</td><td>52* (24-60)</td></tr><tr><td>WOMAC score</td><td>364 (341-389)</td><td>354 (321-389)</td><td>382 (372-407)</td><td>360 (340-400)</td><td>150* (87-217)</td><td>135* (64-238)</td><td>180* (84-218)</td><td>282* (139-318)</td></tr></tbody></table>

<footnote>*P* < 0.05 vs. baseline values (Mann-Whitney-Wilcoxon Test, # P < 0.05 vs. No bDNA values.</footnote>
P-133

THE REDUCTION IN ADRENERGIC AND INFLAMMATORY SERUM MARKERS IN OSTEOARTHRITIS PATIENTS AFTER TREATMENT WITH CHONDROITIN SULFATE/ GLUCOSAMINE HYDROCHLORIDE AND CELECOXIB IS DIFFERENT ACCORDING TO THE PRESENCE OF BACTERIAL DNA IN BLOOD

Zapater P.¹,², Herrero M.³, Martínez H.³, Francés R.²,⁴, Vergés J.⁵, Horga J.F.¹

¹Pharmacology Department, Miguel Hernández University, Alicante, Spain; ²CIBERehd, Instituto de Salud Carlos III, Madrid, Spain;

³Clinical R&D Area, Bioiberica, Barcelona, Spain; ⁴Medicine

Department, Miguel Hernández University, Alicante, Spain;

⁵Osteoarthritis Foundation International, Barcelona, Spain

**Background:** Inflammation in osteoarthritis(OA) has been characterized by infiltration of immune cells and secretion of cytokines into synovial tissues. Adrenergic activity has been associated with subchondral bone loss and increased osteoclast activity in OA. The relationship between activation of adrenergic and immune system with OA progression and treatment response is unknown.

**Objectives:** To assess the adrenergic and immune activation in OA patients treated with Chondroitin Sulfate/Glucosamine Hydrochloride (CS/GH)(Droglican®), Bioiberica, Barcelona) or Celecoxib(CE).

**Methods:** serum samples from patients participants in a 6-month controlled, double blind, randomized clinical trial comparing the analgesic efficacy of CS/GH and CE were analyzed to determine cytokines(IL-2, IL-4, IL-6, IL-10, IL-8, IL-1beta using BD™ CBA immunoassay),catecholamines(noradrenaline, adrenaline and dopamine using ELISA LDN®) and the presence of endotoxin or lipopolysaccharide(LPS; LAL test Lonza®) and bDNA(16S ribosomal DNA-based PCR).

**Results:** Samples from 100 OA patients(age:62 ± 8 yr.; BMI:31 ± 6 kg/m²; 86 females; VAS:73 ± 15; WOMAC:369 ± 43) treated with CS/GH(50) or CE(50) were analyzed. There were no baseline significant differences between the two treatment groups regarding demographics, clinical and experimental variables. Thirty-four patients(n (CS/GH)=17; n(CE)=17) showing bDNA in blood had significantly higher levels of noradrenaline compared with patients without bDNA (1993[1354–3183]vs.1324[738–1899];P = 0.0002).After 6 months, both groups showed a similar reduction in VAS and WOMAC score and serum adrenaline levels independent of presence/absence of bDNA (Table 1).Thirty-three patients showed bDNA presence at the end of the study. Patients with bDNA at 6 months showed reduced serum noradrenaline levels compared with those observed in patients with bDNA at baseline(1993[1354–3183] vs.1561[1174–2193]; P = 0.0325).IL-8 was significantly reduced after 6 months only in patients without bDNA. There were no significant differences between baseline and 6 months samples in the other experimental variables.

**Conclusions:** OA patients show bDNA fragments in blood associated to higher serum noradrenaline levels. 6-month treatment with CS/GH or CE reduces significantly pain and adrenaline levels.IL-8 levels were also reduced except in patients with bDNA fragments in blood. Systemic adrenergic and inflammatory activity in OA patients is influenced by the presence of bDNA in blood and this may be taken into consideration to evaluate severity, evolution and treatments' response.

P-134

TOPICAL PRE-TREATMENT WITH A CAROTENOID FROM MICROALGAE REDUCES CUTANEOUS UVB-INDUCED INFLAMMATION IN HAIRLESS MICE

Rodríguez-Luna A.¹, Ávila-Román J.¹, González-Rodríguez M.L.², Talero E.¹, Zubía E.³, Motilva V.¹

¹University of Seville, Department of Pharmacology, Spain;

²University of Seville, Department of Pharmaceutical Technology,

Spain; ³University of Cádiz, Organic Chemistry, Spain

**Introduction:** Acute exposition to UVB (360 mJ/cm²) induces increase in skin thickness, loss of elasticity and moisture, increase the melanin

production, and promote inflammation. These characteristics are well known as photoaging parameters and play a crucial role in skin cancer induction. Photochemoprevention with natural products is a potent strategy for reduce the development of skin cancer. F-111 is a carotenoid isolated from *Isochrysis galbana* microalgae. The aim of this study was to investigate whether pre-treatment with the carotenoid emulsion is capable to reduce the UVB-induced inflammation in SKH-1 hairless mice.

**Materials:** O/W emulsions were prepared by gradually adding solutions of F-111 (10 mg/ml) or β-carotene (10 mg/ml), used as reference control, and prepared from ethanol absolute to a cold mix excipients. Female SKH-1 hairless mice were topically pre-treated one time per day for three days. Then the animals were exposed to an UVB-exposition (360 mJ/cm²) to induce erythemal response. Animals were sacrificed 48 h later, skin parameters were measured and tissue samples were taken out determination of skin inflammation markers.

**Results:** Our results showed that F-111 emulsion reduced the loss of skin moisture and animals significantly recovered the elasticity comparing with sham; furthermore, melanin index and skin edema were decreased. F-111 also significantly reduced myeloperoxidase (MPO) activity (*P* < 0.001) and inflammatory mediators such as cyclooxygenase-2 (COX-2) (*P* < 0.01) and inducible nitric oxide synthase (iNOS) levels following our experimental model. In addition, we have demonstrated that this compound is involved in the modulation of the nuclear factor-erythroid-derived 2 (Nrf2) (*P* < 0.05) and heme oxygenase 1 (HO-1) (*P* < 0.05).

**Conclusions:** We found that topical treatment with F111-emulsion reduces photoaging parameters and pro-inflammatory skin markers in UVB-irradiated hairless mice. These results indicate that this microalgal carotenoid plays an important role in prevention of inflammatory skin process consequence of sunburn, avoiding important complications including skin cancer.

**Keywords:** Photoaging, Photochemoprevention, UV-irradiation, Microalgae, Skin Inflammation

**Acknowledgements:** Proyecto-ALGALIMENTO, INNPACTO-IPT-2012-1370-060000. Ayudas a la gestión de la investigación V-Plan-Propio-US-PPI2015-1.5. Polfanat-II(P12-AGR-430).

P-135

ANTI-INFLAMMATORY AND SEDATIVE ACTIVITIES OF PEPEROMIA GALIOIDES: IN VIVO STUDIES IN MICE

Wilches I.¹, Jiménez-Castillo P.¹, Cuzco N.¹, Clos M.V.², Jiménez-Altayó F.², Vander Heyden Y.³, Leon-Tamariz F.¹, Vila E.¹,²

¹Faculty of Chemistry, University of Cuenca, Ecuador; ²Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain;

³Department Analytical Chemistry and Pharmaceutical Technology, Vrije Universiteit Brussels-VUB, Belgium

The aerial parts of *Peperomia galioides* popularly known in Ecuador as 'congora blanca' or 'tigresillo' is commonly used in folk medicine in Ecuador for wound healing, auricular inflammation, hysteria, gastrointestinal disturbances, and as sedative in 'susto' illness. Despite its widespread use, few scientific studies support the above claims. Thus, we aimed to confirm the anti-inflammatory and sedative uses of *P. galioides* in folk medicine. The anti-inflammatory activity was evaluated *in vivo* mice models, including croton oil-induced ear edema and myeloperoxidase (acute inflammation); cotton pellet induced granuloma (chronic inflammation) and by *Escherichia coli* Lipopolysaccharide (LPS) induced inflammation (cellular mediators). The sedative activity was evaluated by the pentobarbital-induced sleeping time. Single intraperitoneal administered doses (300 and 600 mg/kg) of *P. galioides* extract significantly inhibited the croton oil-induced ear edema (*P* < 0.001) and myeloperoxidase (*P* < 0.05) activity. Repeated (6 days) administration of the lowest dose of the extract (300 mg/kg) to mice previously implanted with cotton pellets reduced (*P* < 0.05) granuloma formation by 37.8%. Intraperitoneal injection of LPS (20 mg/kg) increased (*P* < 0.001) both plasma nitrites and TNF-α levels that were significantly (*P* < 0.05–0.01) inhibited by the extract. The duration but not the onset of sleeping time, by sodium pentobarbital (40 mg/kg; i.p.), was dose-dependently enhanced by 300 and
600 mg/kg of the extract. Our results demonstrate that *P. galioides* has anti-inflammatory as well as sedative activities in mice, which validates some of its traditional uses.

**Keywords:** anti-inflammatory activity, sedative activity, *Peperomia galioides*

This work was funded by: DIUC (Cuenca University); VLIR-UOS project 'Pharmacological Characterization of Medicinal Plants from the South of Ecuador'. E.V. by Proyecto Prometeo, Secretaria de Educación Superior, Ciencia, Tecnología e Innovación de la República del Ecuador.

## P-136
HEPATOPROTECTIVE EFFECT OF CROTON HYPOLEUCUS FOLLOWING THIOACETAMIDE-INDUCED NECROSIS IN RATS

Urrutià-Hernández T.A., Velázquez-González C.¹, De la O-Arciniega M.¹,²,³, Morales-González J.A.³, Castañeda-Ovando A.¹,²,³, Güemes-Vera N., Benedí J.², Sánchez-Muniñ F.J.², Bautista-Avila M.¹

¹Instituto de Ciencias de la Salud, Universidad Autónoma del Estado de Hidalgo, Pachuca, Hidalgo, Mexico; ²Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain; ³Escuela Superior de Medicina, Instituto Politécnico Nacional, México D.F., México

**Introduction:** *Croton hypoleucus* (Ch) is used by traditional medicine in Central Mexico for inflammatory processes or to treat respiratory illnesses like cough. However, scientific evidence does not exist in any literature to corroborate the claim of therapeutic success of Ch in these targets or any other. In the present report the effect of Ch extract were studied in reference to postnecrotic liver damage induced in rats by thioacetamide (TA).

**Material & Methods:** Rats, pretreated with a single dose of *Croton hypoleucus* ethanol extract (300 mg/kg) every 24 h for four days, were intraperitoneally injected with TA (6.6 mmol/Kg) at fourth day of treatment. Samples of blood and liver were obtained at 24 h following TA intoxication and parameters related to liver damage like AST and ALT were carried out in blood using well established protocols and methods. Results

The results showed that the pre-treatment with crude ethanol extract significantly reduced liver damage. *Croton hypoleucus* decreased liver injury by 40% and 30% for ALT and AST (biochemical markers of liver damage) respectively at 24 h the peak of maximum regeneration. Also the LD (50) of the plant species was obtained and this was greater than 5000 mg/kg (p.o.).

**Conclusion:** The data obtained indicate that the crude extract of *Croton hypoleucus* has hepatoprotective activity. However, further investigation on the mechanism of the hepatoprotective effect of the plant species needs to be carried out.

**Keywords:** croton Hypoleucus, hepatoprotective effect, thioacetamide

## P-137
EFFECTS OF OLIVE LEAF EXTRACT IN HIGH-FAT DIET-FED MICE: IMPACT ON VASCULAR DYSFUNCTION

Diez-Echave P.¹, Vezza T.¹, Algieri F.¹, Rodríguez-Nogales A.¹, Garrido-Mesa J.¹, Toral M.², Romero M.², Sanchez M.², Rodríguez-Perez C.³,⁴, Gálvez J.¹

¹Department of Pharmacology, CIBER-EHD, ibs.GRANADA, CIBM, University of Granada, Spain; ²Department of Pharmacology, CIBER-EnfermedadesCardiovasculares, ibs.GRANADA, CIBM, University of Granada, Spain; ³Department of Analytical Chemistry, University of Granada. Avda. Fuentenueva s/n, 18071 Granada, Spain; ⁴Research and Development of Functional Food Centre (CIDAF), PTS Granada, Avda.delConocimiento s/n, EdificioBioregión, Granada, Spain

**Introduction:** Different studies have reported the beneficial effects of the olive (*Olea europaea*) leaf extract (OLE)in experimental models of

metabolic syndrome, being this activity ascribed to the ability of its phenolic compounds, like oleuropeoside, to exert immunomodulatory, hypoglycemic and hypocholesterolemic activities. However, little information is available about its impact on obesity-associated vascular dysfunction.

**Aim:** To evaluate the effects of a well defined olive leaf extract in high fat diet (HFD)-induced obesity in mice, and to investigate the underlying mechanisms involved in the beneficial effect, with special attention to vascular dysfunction.

**Methods:** Male C57BL/6J mice were divided into several groups: control, control-treated, obese and obese-treated with OLE (1, 10 and 25 mg/kg/day) or Metformin(250 mg/kg/day).Control mice received a normal chow diet(NCD), whereas obese mice were fed(HFD).The treatment was followed for 6 weeks, and animal body weight periodically assessed. At the end of the experiment, metabolic plasma analysis was performed, including lipid profile, as well as glucose and insulin levels. Additionally, the HFD-induced inflammatory status was studied biochemically in liver and fat, by determining the RNA expression of different inflammatory mediators and markers of intestinal epithelial barrier function by qPCR. Also, endothelial dysfunction in aortic rings was also evaluated.

**Results:** OLE administration reduced body weight gain, basal glycaemia and insulin resistance, and showed improvement in plasma lipid profile when compared with HFD-fed mice. The extract significantly ameliorated the HFD-induced altered expression of key adipogenic genes, like PPARs, adiponectin and leptin, in adipose tissue. Furthermore, the extract reduced the RNA expression of TNFα, IL-1β, IL-6 in liver and adipose tissue, thus improving the inflammatory status associated to obesity. Additionally, the extract reversed the endothelial dysfunction observed in the aortic rings of obese mice.

**Conclusion:** OLE exerts beneficial effects in HFD-induced obesity in mice, which was associated to an improvement in plasma and tissue metabolic profile, inflammatory status and vascular dysfunction.

**Keywords:** experimental obesity, olive leaf extract, polyphenols, endothelial dysfunction

## P-138
HEMP (CANNABIS SATIVA L.) SEED OIL AS A SOURCE OF BIOACTIVE COMPOUNDS FOR THE TREATMENT OF FIBROMYALGIA-RELATED SYMPTOMS

Montserrat-de la Paz S.¹,², García-Giménez M.D.¹, Bermúdez B.³, Marín-Aguilar F.¹, Ángel-Martín M.¹, Quílez A.¹, de la Puerta R.¹, Fernández-Arche A.¹

¹Department of Pharmacology, School of Pharmacy, University of Seville, Spain; ²Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa, CSIC, Spain; ³Department of Cell Biology, Faculty of Biology, University of Seville, Spain

**Introduction:** This study was designed to evaluate the effect of hemp (*Cannabis sativa* L.) seed oil (HSO) on an experimental mouse model of fibromyalgia syndrome (FMS) induced by intermittent cold stress (ICS) and to evaluate the anti-inflammatory effect of the bioactive components in HSO.

**Material and Methods:** HSO phytochemical characterization was carried out by GC-MS and HPLC techniques. Sixty mice (C57BL/6J) were used for the experiments and randomized into six groups (n = 10): healthy control group and ICS group, were fed a standard diet, and the remaining groups received a standard diet supplemented with the respective dietary fats (10%, w/w): ICS+sunflower oil, ICS+refined olive oil, ICS+hemp seed oil, and ICS+fish oil. Freshly human monocytes were used to analyse the effects of phytol and cycloartenol (10–100 mM) on inflammatory response using FACS analysis, RT-qPCR, and ELISA procedures.

**Results:** The lipid profile showed that linoleic, α-linolenic, and oleic were the most abundance fatty acids. A yield (1.84–1.92%) of unsaponifiable matter was obtained and the most interesting compounds were: β-sitosterol, campesterol, phytol, cycloartenol, and γ-tocopherol. In ICS-induced FMS, HSO consumption improved mechanical and thermal allodynia and mechanical hyperalgesia (Paw
pressure, hot plate, and tail immersion tests) and improved behavioral
changes related to cognitive disturbances, anxiety, and depression (hole
board, traction, and evasion tests). Phytol and cycloartenol skewed the
monocyte plasticity towards the anti-inflammatory non-classical
CD14⁺CD16⁺⁺ monocytes macrophage phenotype and reduced the
inflammatory IL-1β, IL-6, and TNF-α production.

**Conclusions:** This study is an important contribution for *Cannabis sativa* L. valorization as a source of bioactive compounds contributing to research novel applications (e.g., fibromyalgia syndrome) for hemp seed oil in the food, pharmaceutical, cosmetic, and other non-food industries.

**Keywords:** Hemp seed oil; Cannabis sativa; Fibromyalgia; Inflammation

P-139
HOW DID THE PRESS APPROACH THE DISCOVERY AND
USE OF SULPHONAMIDES DURING THE 20TH CENTURY?

Martín-Gonzalvo N.¹, López A.¹, Serés E.¹, Bosch F.¹,²

¹Esteve Foundation, Barcelona, Catalonia, Spain; ²Department of
Experimental and Health Science, Universitat Pompeu Fabra,
Barcelona, Catalonia, Spain

**Introduction:** The advent of the sulphonamides changed the treatment of infectious diseases during the 20th century. The scientific literature clearly records the development and use of these drugs; however, how the mass media transmitted these advances has not been extensively studied. We aimed to quantitatively and qualitatively evaluate reports concerning the discovery and use of sulphonamides in the press during the 20th century.

**Methods:** We searched for news reports on sulphonamides published between 1908 and 1979 in *The New York Times* (NYT), *The Times of London* (TTL), and *La Vanguardia* (LVG). The selected reports were quantitatively analyzed and categorized in function of their year of publication and other general features. Reports considered scientifically informative were analysed qualitatively using the Oxman Index of Scientific Quality (ISQ).

**Results:** We retrieved 2101 reports; 788 (37.5%) were excluded (duplicate reports, ads and newspaper indexes). Thus, 1313 (62.5%) reports were selected for analysis: 986 (75.1%) from NYT, 193 (14.7%) from TTL, and 134 (10.2%) from LVG. Of these, 812 (61.8%) reports were published between 1938 and 1949. The number of reports published peaked in 1943 (n = 142, 10.8%). Most reports (n = 982, 50.1%) dealt with general aspects about sulphonamides. Reports on pharmacology aspects (n = 523, 26.7%) dealt predominantly about clinical studies (n = 197, 26.4%) and route of administration (n = 147, 18.3%) issues. The qualitative analysis of the 20 (1.5%) reports considered informative (9 from NYT, 7 from TTL, and 4 from LVG) yielded a mean ISQ score of 24.1 (SD=3.8; range 17.0–30.0), equivalent to 6.1 on a 10-point scale.

**Conclusions:** The number of news reports published in the press adequately reflects the development of sulphonamides. A low percentage of reports were considered truly disseminative, and the scientific quality of these was acceptable.

**Keywords:** health communication; history; mass media; sulphonamides

P-140
ATTRACTIVE KEY HISTORICAL FACTS AS A TOOL TO
TEACH PHARMACOLOGY

López A.¹, Martín-Gonzalvo N.¹, Serés E.¹, Bosch F.¹,², Baños J.E.²

¹Esteve Foundation, Barcelona, Catalonia, Spain; ²Department of
Experimental and Health Sciences, Universitat Pompeu Fabra,
Barcelona, Catalonia, Spain

**Introduction:** Pharmacology is an important subject in many university undergraduate programmes. Different pedagogical strategies, from

lectures to problem-based learning sessions, can be used for teaching
pharmacology. This study aimed to select key historical information
related to the first antimicrobials to enhance the teaching of pharmacol-
ogy.

**Methods:** We examined review articles and material in pharmacology textbooks related with the first antimicrobials: arsphenamine, sulphonamides, and streptomycin. Subsequently, we searched *The New York Times*, *The Times of London*, and *La Vanguardia* for news published on these drugs between 1908 and 1979 to identify key historical information that could be attractive for teaching. Finally, we wrote a brief summary of each selected issue.

**Results:** A review of 50 scientific articles, 82 books, and 3,078 news articles yielded 28 key historical issues: 13 on arsphenamine, 9 on sulphonamides, and 6 on streptomycin. Examples include the mistaken attribution of the discovery of arsphenamine to Paul Ehrlich's Nobel Prize; U.S. President Franklin Roosevelt's son's miraculous recovery thanks to sulphonamides; and sexist discrimination against Elizabeth Bugie in assigning credit for the discovery of the streptomycin. Other selected facts addressed issues such as new pharmaceutical dosage forms, drug toxicity, new indications for drugs, patents, commercial interests, and the use of drugs during war.

**Conclusions:** Reviewing historical information related to drugs makes it possible to identify material that can enhance the teaching of pharmacology. Interesting anecdotes about arsphenamine, sulphonamides, and streptomycin can serve not only for teaching antimicrobials but also for pharmacology in a much broader sense.

**Keywords:** antimicrobials; history; pharmacology; teaching

P-141
USING CINEMA FILMS AS AN INNOVATIVE
EDUCATIONAL STRATEGY TO CREATE MEANINGFUL
LEARNING IN PHARMACOLOGY TEACHING

Cabanillas-Sáez A.¹, Acevedo J.A.²

¹Department of Pharmacology, Faculty of Biological Sciences, University of Concepción, Concepción, Chile; ²Department of General Studies, Faculty of Education and Humanities, University of Bío-Bío, Concepción, Chile

The teaching of Pharmacology for fourth year students of Biochemistry represents an interesting challenge for the teachers in the field because it is taught simultaneously with vocational training subjects that demand a lot of time and energy from students. The application of the *Honey-Alonso Learning Styles Questionnaire (CHAEA)* evidenced that these students' predominant learning styles were the reflexive-theoretical modes. Since one of the activities that strengthens the learning process in this group of students is the analysis of films or videos on specific subjects, five popular films related to different aspects of Pharmacology were chosen and exhibited as part of the course syllabus in a films series called 'Movie Mornings in Pharmacology'. Each film session included a complete technical file and a list of expected learning outcomes to guide the students in the analysis of the film. At the end of the course, the students had the opportunity to write an analytical essay about these films or to give an oral presentation including their own conclusions regarding each movie. This innovative teaching tool implemented in the subject of Pharmacology has allowed the students to integrate the basic knowledge of the discipline with ethical and social issues involved in the use of medicines, develop a critical attitude towards possible advantages and risks of pharmaceutical drugs commonly used by the community and increase their motivation regarding the subject and the program. It has also provided teachers with the opportunity to contextualize the contents of the subject, to implement alternative assessment methods, to enhance teacher-student interactions and to contribute effectively to create and promote meaningful learning experiences regarding pharmacology.

**Keywords:** pharmacology, cinema, meaningful learning
P-142

ACTIVE LEARNING: ALIGNMENT OF ASSESSMENT AND OBJECTIVES

Rangachari P.K., Nastos S.

BHSc (Honours) Program/Medicine

Effective learning in any format needs close alignment with objectives and assessments. This is particularly so in courses that foster active learning since these make more demands on students. Here we report on our 11-year experience in a first-year introductory undergraduate course in cellular biology in a large class setting (160–255 students). In term 1, interactive didactic sessions gave students a broad overview of cellular signalling (gene expression, synthesis, storage, release, effects and termination of responses to specific signalling molecules: histamine, acetylcholine, catecholamines, prostaglandins, nitric oxide and steroids). Students learnt not just the ‘facts’ but the ‘way’ to those facts, with an emphasis on approaches used to gather them. In term 2, students were expected to go beyond the mere facts to explore their construction, demonstrate creativity in transferring learning to novel situations, as well as flexibility in responding to challenges. A range of assessment exercises that fostered active engagement were used: (i) process-oriented problem-solving exercises; (ii) abstract writing exercises to get students to recognise key elements of peer-reviewed publications; (iii) a project to explore key discoveries in cell signaling through the Nobel archives; (iv) a project to explore the molecular basis of specific sins (greed, lust, anger, sloth); and, (v) a project to design problem-solving exercises and frame acceptable solutions and leave these as legacies for future classes. The course received high ratings from 813 students who felt that it provided them a valuable learning experience (9.0 ± 1.6 on a 10-point scale).

P-143

PREPARING PHARMACY STUDENTS TO PRACTICE: ESPECIALICED REPORTS AND ORAL COMMUNICATION ACTIVITIES

Recio M.C., Ferrándiz M.L., Merino V., Dualde E., Merino M.

**Introduction:** Students in the 5th year of the Pharmacy degree must complete the compulsory subject 'Clinical Pharmacy and Pharmaceutical Care'. Students must acquire the skills and ability to identify, integrate and apply the knowledge in the medical and pharmaceutical sciences to resolve through communication, information and education to the patient and other health professionals. Seminar sessions are one of the best settings to interact with the students in small groups reinforcing the transmission of these types of skills.

**Method:** Teacher presents the types of seminar they can perform: 1) Medication reconciliation at transitions in care and active dispensing of emergency contraception pills prescription; 2) Drug selection by means the System of Objectified Judgement Analysis method, and 3) Health education through a service-learning (S-L) project. Students, in teams of four, choose the activity they prefer to work with and prepare the necessary material. Following, each working team, with the help of the teacher and the valuable sources uploaded to the virtual platform, prepare a documented analysis of their cases or the activities and materials to perform the S-L project. The latter is done outside the classroom, in a social institution. Finally, all the groups give the dossier to the teacher and share and discuss their works with their colleagues.

**Results and Discussion:** Evaluation was carried out with a survey about the quality of the presentation and debate, and the usefulness of the activity towards their future role as pharmacists. The students become aware of the role of the pharmacist to reduce the number of medication errors and improve the quality of care. Moreover, these activities promote students' communication skills and their ability to search information and produce well-reasoned written reports concerning therapeutic issues, addressed to either patients or health professionals.

P-144

MOTIVATIONAL TOOLS TO ENCOURAGE STUDENTS TO BECOME ENGAGED IN A PHARMACOLOGY SUBJECT

García Vieitez J.J., Fernández Martínez M.N., Díez Suárez A., Diez Liébana M.J., Díez Láiz R., Sierra Vega M., Sahagún Prieto A.M.

Department of Biomedical Sciences, IBIOMED, University of Leon, León, Spain

Teachers play a crucial role in students' motivation, which is seen as one of the most important and determinant factors for academic success as well as promoting long life learning. The aim of this survey was to implement strategies for active learning in an elective subject of the Degree in Physiotherapy (Drugs and Sport), in order to improve students' motivation for the subject and encourage their autonomous learning.

Two enforcement activities (reading of news related to the subject and questionnaires) were used. A satisfaction survey was also provided to students to value their opinion. 32 students took part voluntarily in the innovative strategy (94.1% of those enrolled in the subject): 75% were female and 25% male. Four articles on doping and its adverse consequences were read and commented at the end of the corresponding topic. Four on line questionnaires were provided via Moodle in order to self-assess the knowledge of the contents. They had to be answered in a limited period of time, and three attempts were allowed.

Scores achieved in quizzes were high, and response times were progressively reduced, as well as the number of attempts needed by the participants. Final scores in the subject were slightly higher than those enrolled in the previous academic year (no significant differences, Mann-Whitney U test, p < 0.05). In satisfaction survey most students considered that the number of articles and questionnaires was suitable, and also their degree of difficulty. Moreover, both strategies have improved their interest and motivation for the subject, and they were highly satisfied with them.

P-145

RUBRIC-BASED SCORING IN A HANDS-ON SESSION OF VETERINARY PHARMACOLOGY

Díez Láiz R., Sierra Vega M., García Vieitez J.J., Diez Liébana M.J., Fernández Martínez M.N., Rodríguez Lago J.M., Sahagún Prieto A.M.

Department of Biomedical Sciences, IBIOMED, University of Leon, León, Spain

At present, the assessment at University is undergoing profound shift from traditional evaluation of factual knowledge towards assessment for learning and competences. The use of rubrics could improve the quality of the evaluation made. The objective of this study was to develop a skill assessment method in a hands-on session conducted with mice in the subject Veterinary Pharmacology.

A specific analytic rubric was designed to evaluate, by means of performance criteria and indicators, the acquisition of skills related to the pharmacological action of the drug used in the practical session, their ability to work in team, a respectful treatment of animals, or their capacity of analysis and synthesis. 132 students (74.2% female) were evaluated with the rubric. Skills defined for this practical session were reached by all students. They acquired 76.0 ± 5.6% of those competencies previously set. Female students were capable of acquiring 78.7 ± 5.8% of skills, whereas male students showed a significantly lower level (76.2 ± 4.9%, Mann-Whitney U test, P < 0.05). Competences addressed to work in team and handling of animals achieved the highest scores, and those related to capacity of analysis and summary of results obtained the lowest values.

Rubrics are a suitable and useful instrument to assess complex performances and provide evidence of student achievements, although it is necessary the commitment of the entire teaching staff who collaborates in a certain subject.
# P-146
## STUDENTS OF THE PRIMARY EDUCATION DEGREE, AS THE NEW PLAYER IN HEALTH AND NUTRITION PROMOTION

Martín Rubio M.E.¹, Navarro P.D.¹, Montserrat-de la Paz S.²,³,
Bermúdez B.¹

¹Department of Cell Biology, Faculty of Biology, University of Seville, Spain; ²Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa, CSIC, Spain; ³Department of Pharmacology, School of Pharmacy, University of Seville, Spain

**Introduction:** Primary Education Degree (PED) students are the future teachers for children aged 6 to 11 years old. As professors, it is our responsibility to encourage these students to achieve both scientific and social skills. This is remarkable if we consider that they must develop teaching strategies concerning health and nutrition. The main goal of this proposal is to improve scientific training concerning lipid metabolism and diet of the future PE teachers.

**Material and Methods:** The students of the first year of PED are distributed into groups. They choose several 'key words' related with the metabolism of lipids present in our diet to prepare a scientific project during classroom sessions, to finally explain their conclusions in class. We also give them a test including questions about this topic, before and after the activity. A control group of students do not participate in any project but work both pre and post tests. Professors will be available for additional tutorial sessions to support student's activity out of the classroom.

**Results:** After evaluating the scientific projects and the answers of the tests, we can conclude that the participants reveal a significant increase of the level of scientific knowledge concerning lipid effects in our diet, in contrast to the students control group. Moreover, one of the questions of the tests let them propose teaching activities in their future classes, and it is remarkable that they have many and very interesting ideas. We must highlight the level of implication of the students in this proposal.

**Conclusions:** We provide evidence that new teaching strategies should allow the students become the main actors of the learning process. Our students have improved both their knowledge concerning nutrition and their skills in scientific education research. It is also important to develop teaching proposals which are quite appropriate with the needs of our future citizens.

# P-147
## THE LAST FRONTIER: INTER PROFESSIONAL LEARNING EVENTS BETWEEN DENTISTRY AND PHARMACY

Ferrie L.J., Sturrock A.

School of Biomedical Sciences, Newcastle University, Newcastle upon Tyne, UK: School of Pharmacy and Pharmaceutical Sciences, University of Sunderland, Sunderland, UK

Undergraduate students from across a range of healthcare professional courses often struggle to apply fundamental pharmacological principles to their clinical practice. Problem-based or case-study centred curriculums provide some resolution to this problem but often lack the depth of detail and complexity needed to showcase how basic pharmacokinetic and pharmacodynamic (PK/PD) understanding should be used to address drug safety issues. Interprofessional (IPL) learning, as a complementary teaching method, not only supports students understanding of pharmacological decision making processes but also prevents the development of a 'silo mentality' by encouraging greater professional teamwork and communication (Harden, 2000). Both Wilson and Soni (2016) and Pemberton (2014) argue that greater focus should be placed on the benefits of encouraging IPL links between dentistry and pharmacy to support patient safety.

This pilot project developed two unique IPL workshops which allowed students to explore, collaboratively, how PK/PD knowledge can be used to address drug interaction issues in patient's prescribed anti-coagulant or antibiotic therapy.

The study aimed to determine if students would further their knowledge of drug mechanisms of action through discussion with other professional groups. 23 students attended the 3 hour session (18 pharmacy, 5 dentistry) and completed a perception survey before and after the workshop to rate their therapeutic knowledge and understanding of healthcare professional roles. Students reported a 52% and 30% increase in their antibiotic and anti-coagulant therapeutic knowledge as a result of the workshop. Students also reported that their appreciation of the practice decisions of other healthcare professionals increased by 18% post-workshop. 82% of students agreed that the workshop helped them to understand how to work with other professionals to resolve clinical practice issues.

The results from this study show how IPL events can provide an enriched pharmacology curriculum, enhance therapeutic knowledge and provide a unique learning opportunity for undergraduate students.

**Keywords:** Interprofessional learning

# P-148
## RATIONAL PHARMACOTHERAPY IN THE ASSESSMENT OF CLINICAL COMPETENCE USING AN OBJECTIVE STRUCTURED CLINICAL EXAMINATION 'OSCE'

Blanco-Reina E.¹, Bellido I.¹, Lucena M.I.¹, Cabello M.R.¹, García-Arnés J.A.¹, Slim M.¹, Sanabria J.¹, Valdivielso P.²

¹Servicio de Farmacología Clínica y Departamento de Farmacología, Instituto de Investigación Biomédica de Málaga (IBIMA), Hospital Universitario Virgen de la Victoria, Universidad de Málaga, Málaga, España; ²Comisión ECOE Facultad de Medicina, Universidad de Málaga

Drugs are the mainstay of medical treatment. Pharmacology training for most medical students concentrates more on theory than on practice, but in clinical practice it must be known how to select the best option for the particular patient. On the other hand it is necessary to encourage innovation in medical education. The examination should meet three main criteria: validity, reliability and close to real practice. The Objective Structured Clinical Examination 'OSCE' was designed to assess student's clinical competence. The aim of this work has been to assess the results of the implementation of a pharmacotherapy station in the OSCE for sixth-year Medicine students.

**Methods:** A multidisciplinary team of clinical specialists organized an OSCE including 20 stations. There were stations with actors, patient simulators, radiological images and others. In the pharmacotherapy station students were presented a clinical case, laboratory data and medication used. They had to optimize the therapeutic plan and fill in on a model of common electronic prescription. The clinical case was a patient with hypertension, diabetes and dyslipidemia. The patient presented lower back pain, persistent dry cough, microalbuminuria and high blood pressure levels. We established 10 items that assessed clinical judgment and therapeutic management plan.

**Results:** 150 students were examined in the hospital setting (Málaga). The average rating of the whole stations ranged between 4.2 and 8.9. Pharmacotherapy station reached 7.48 (range 1.5–10), with Gaussian distribution. The easiest items were pharmacodynamic interaction and side effects. However, the correct choice of analgesia was the most difficult skill. There was a very good level of discrimination.

**Conclusions:** We have found that the Therapeutic OSCE is feasible and useful to assessing prescribing competences. Given that prescribing requires the acquisition of knowledge, skills and attitudes, we think that the incorporation of clinical pharmacology in this final examination is essential.

**Keywords:** OSCE; medical education; rational prescribing skills; clinical pharmacology
P-149

DOES THE DESIGN AND DEVELOPMENT OF OBJECTIVE STRUCTURED CLINICAL EXAMINATIONS BY UNDERGRADUATE STUDENTS OF PODIATRY DEGREE IMPROVE THEIR LEARNING AND ASSESSMENT SKILLS ABOUT MEDICINES?

Inmaculada B.E., Encarnación B.R., Victoria B.E.M., Antonio G.A.J., Aurelio G.L.

Dep Farmacology, School of Medicine, University of Malaga, Spain;
Dep Farmacology, School of Medicine, University of Malaga, Spain;
S. of Anaesthesia, Regional Carlos Haya-Mothers and Children
Section, Malaga, Spain; Dep Farmacology, School of Medicine,
University of Malaga, Spain; S. of Anaesthesia, Virgen de la Victoria
University Hospital, Malaga, Spain

We aim to evaluate the impact of designing, developing and presenting
Objective Structured Clinical Examinations (OSCEs) by undergraduate
students on the Pharmacology course of Podiatry Degrees in their
learning of medicines uses.

We carried out a three-year prospective study in which students on the Pharmacology course of Podiatry Degree were invited to voluntarily design and perform an OSCE. Each group (4–5 students) reports a clinical situation/problem involving medicines for 10 min max. Clinical histories, cameras, a mobile-phone’s video editor, photos, actors, dolls, simulators or whatever they may use was allowed. After each OSCE performance the other students of the class were encouraged to ask questions and they should criticize the work of each OSCE-group indicating the possible committed errors in both the approach and development of each OSCE. The results in the final exam of the students of the three previous courses without OSCEs were used as historical controls for comparison purposes.

This study improved the learning of pharmacology and medicines uses of undergraduate students on the Pharmacology course of Podiatry degree (N = 189, 59.8% female, 19 ± 2.7 years old) and allowed us to make 43 OSCEs showing a clinical situation or a clinical problem. The students did not spend a lot of time making the OSCEs (31.7 ± 14.7 h). Spoken participation and communicative skills of the students in class increased as they were developing the OSCEs. The percentage of success in the final subject assessment questions was significantly higher in the OSCEs related question (92.5%) compared with the OSCEs un-related question (68.2%) and with the historical controls 76.5 (P < 0.05). The percentage of students satisfied with this way of studying pharmacology was 92.7%.

Objective Structured Clinical Examinations designed and performed by
undergraduate students on the Pharmacology course of Podiatry
Degree improved their knowledge about medicines use and their final
assessment results.

P-150

IMPLEMENTATION AND EVALUATION OF AN ONLINE SELF-ASSESSMENT SYSTEM TO IMPROVE STUDENT PERFORMANCE IN PHARMACOLOGY

Moreno L.¹,², Cogolludo A.¹,², Barrigon S.¹, Pérez-Vizcaíno F.¹,²

¹Department of Pharmacology, School of Medicine, Universidad Complutense de Madrid; ²CIBER of Respiratory Diseases (CIBERES)

Active learning tools have been shown to enhance student learning and promote knowledge retention in several disciplines. The aims of the present study were to incorporate a self-assessment tool in the virtual platform Moodle and to evaluate its potential to improve student's performance in a Pharmacology course.

Students enrolled in Pharmacology (3rd course of Medical Degree, UCM) were the potential participants of this study (n = 360). A test bank of true/false questions covering 9 topics addressed during each semester was made available to students through Moodle. Using a crossover design, participants had unlimited access to self-assessment quizzes (10 questions/quiz) for 12 days after finishing the theory

lessons. The week before the formal assessment, the participants were
offered two full test attempts (85 questions each). Results on student's
performance on both optional and formal assessments were collected
from Moodle and exported to perform statistical analysis (2-way
ANOVA; *P* < 0.05). Adherence to protocol ('active participants') was
defined as completing 75% of self-assessments and the formal assess-
ment in each semester.

During the first semester, 41.8% of the students completing the mid-
term formal assessment (n = 153) showed adherence to the protocol.
Active participants obtained higher marks (6.04 ± 1.99) than students
in the control group (4.88 ± 2.33) in the topics included in the self-
assessment quizzes (P < 0.001). These differences remained significant
despite including the non-adherent participants in the analysis
(5.43 ± 2.27; P = 0.019). However, during the second semester, the
participation dropped to 22.9% of the students completing the final
formal assessment (n = 109) and only active participants obtained sig-
nificantly higher marks in the relevant topics as compared to those in
the control group (7.38 ± 1.68 vs. 6.49 ± 1.92; P = 0.017).

Although the low participating rate suggest that this system has a lim-
ited value to engage students in learning activities on a daily basis,
offering this tool might improve the performance of medical students
in pharmacology courses.

P-151

NEW ACADEMIC PERSPECTIVES IN TEACHING PHARMACOLOGY AND CLINICAL THERAPEUTICS: AN INTERNATIONAL SERVICE LEARNING EXPERIENCE

Cabello M.R.¹, Barquín J.², Lucena M.I.³

¹Dpto. Farmacología. Facultad de Medicina. Universidad de Málaga, Málaga, España; ²Departamento de Didáctica y Organización Escolar. Facultad de Ciencias de la Educación. Universidad de Málaga, Málaga, España; ³Servicio de Farmacología Clínica, Instituto de Investigación Biomédica de Málaga (IBIMA), Hospital Universitario Virgen de la Victoria, Universidad de Málaga, CIBERehd, Málaga, España

**Introduction:** Service-learning (SL) is an educational method that is expanding the involvement of universities into their neighbouring communities. It also tends to promote civic and moral development of students. Service-learning can be defined as ‘service performed by students, aimed at attending to a real need of the community, and oriented in an explicit and planned way to enhance the quality of academic learning.’ (1)

Furthermore International Service Learning (SL) has enabled health professional students the opportunity to provide healthcare, under the direction of a trained faculty, to underserved populations in developing countries.

**Objective:** To verify the usefulness of International SL experience in learning pharmacology, to support or not the design of a new pharma-cology subject based on SL.

**Methods:** We designed a cooperation project with ACOES-Honduras to develop a training experience for students of medicine. This study is based on the qualitative analysis of the final reports of 8 participants.

**Results:** The analysis was focused on 27 transversal and 19 specific competences that cover areas of practice and others related to Pharmacology. In the case of transversal competences, around 70% were covered in the activities carried out. Specific competences were covered up to 75%. Volunteer narratives provided an image of the experience that showed the effectiveness of this type of program, both in increasing sensitivity and attention to diversity and in the implementation of 'theoretical' training, and the original response to unforeseen situations.

**Conclusions:** SL was found to be an effective method to acquire personal and professional competences and skills in the area of pharmacology demand by our present society. Hence we will suggest this methodology in the design of a new subject: 'Pharmacotherapeutics in disadvantaged environments'.

(1) Tapia, MN, et al. (2006). 'Service-Learning in Argentina Schools.' Casey, Karen McKnight, et al. (eds.).
P-152
PEER INSTRUCTION IN VETERINARY PHARMACOLOGY:
A PRELIMINARY EXPERIENCE

Clos M.V.

Pharmacology, Therapeutic and Toxicology Department, Veterinary Faculty. Institut de Neurociències. UAB - Spain

Peer Instruction (PI) is a student-centered approach in which the class time is used for learners to construct concepts rather than for instructors to lecture. Differing from those in traditional lectures, PI students requires to read and complete assignments before attending class (1). There are different research data that supports the effectiveness of PI over more traditional teaching methods, such as pure lecture (2). We used PI for first time in our Pharmacology cours, specifically we applied this teaching methodology in the Pharmacology of Hemostasis. The class time was focused on two different ConcepTests: Antiplatelet agents and Anticoagulants/ Fibrinolytics drugs with a previous 5 min minilecture. The number of questions were 6 and 8, respectively, according to the main key points of each section. Each question had four options and one or two of them were correct.

For this purpose the free platform SOCRATIVE was used, and students, through their laptops or cell phones, answered the test based on what they have learned during the preinstructional reading assignments. In the first assessment of ConcepTest the global incorrect answers were between 64-56%. Consequently students discussed answers with others classmates and some general questions were discussed by the instructor with the class as a whole. After, students revoked the first ConcepTest and the number of incorrect answers decreased up to 10-7%. Then we moved to the next ConcepTest. Similar results were observed with the ConcepTest of anticoagulant/fibrinolytic drugs.

Developing good ConcepTests certainly takes a great deal of effort, however during the development of this inverted class students showed a high degree of involvement. We obtained a positive feedback from them. The only criticism was regarding the time spent in preparing the subject.

(1) E. Mazur, Peer Instruction: A User's Manual (Prentice-Hall, Upper Saddle River, NJ, 1997).

(2) P Zhang, L Ding and E Mazur. PRPER 113, 010104 (2017).

P-153
EPIGENETIC MECHANISMS UNDERLYING NEUROPATHIC PAIN IN RATS

Francés R.<fnref n="1" />,<fnref n="2" />, Mata-Garrido J.<fnref n="2" />,³, Lafarga M.<fnref n="2" />,³, Berciano M.T.<fnref n="2" />,³, Tramullas M.<fnref n="1" />,<fnref n="2" />, Hurlé M.A.<fnref n="1" />,<fnref n="2" />

¹Departamento de Fisiología y Farmacología, Facultad de Medicina, Universidad de Cantabria, Santander, Spain; ²Instituto de Investigación Valdecilla (IDIVAL), Santander, Spain; ³Departamento de Anatomía y Biología Celular, Facultad de Medicina, Universidad de Cantabria, Santander, Spain

Neuropathic pain (NP) subsequent to lesions or diseases affecting the somatosensory nervous system is a prevalent and debilitating chronic syndrome highly refractory to current analgesics. The development and maintenance of NP encompass long term pathological plasticity in the nervous system.

**Objective:** To investigate the involvement of epigenetic mechanisms in NP pain establishment.

**Methods:** Neuropathic pain was induced to rats by sciatic spared nerve injury (SNI). Mechanical allodynia was assessed with von Frey monofilaments. At the moment of maximal allodynia (10 days post-SNI) lumbar (L3, L4, L5) spinal dorsal horns (SDH) and dorsal root ganglions (DRG) were obtained and processed for quantitative PCR or immunofluorescence.

**Results:** The transcript levels of DNA methyltransferase-3b (DNMT3b) and histone deacetylases (HDACs) 4 and 9 were significantly up-regulated in the SDH and DRG from neuropathic rats. In

parallel, both structures exhibited increased methylation at cytosine in CpG islands, assessed by immunofluorescence with Ab to 5-methylcytosine, as well as methylation of histone H3K9, assessed by Ab to H3K9me3.

**Conclusions:** Epigenetic modifications including DNA and histone methylation suggest transcriptional repression in pain-relevant structures associated to neuropathic pain development. (Supported by SAF2016-77732-R).

P-154
SENSITITIZATION OF TRPV1 NEURONS BY PGE2: LOST OF SENSORY SELECTIVITY FOR NOXIOUS HEAT AND PARTICIPATION ON MECHANICAL HYPERALGESIA

Ruiz-Cantero M.C.<fnref n="1" />, Montilla-García Á.<fnref n="1" />, Perazzoli G.<fnref n="2" />, Sánchez-Fernández C.<fnref n="1" />, Baeyens J.M.<fnref n="1" />, Cobos E.J.<fnref n="1" />,³

**Introduction:** Peptidergic (TRPV1-expressing) C-fibers code for noxious heat under normal conditions, but not for mechanical stimulus. Prostaglandin E2 (PGE2) sensitizes peptidergic C-neurons by TRPV1 phosphorilation through protein kinase A (PKA) activity, leading to heat hyperalgesia, whereas glial cell derived neurotrophic factor (GDNF) sensitizes other neuronal types (which do not express TRPV1), leading to mechanical hyperalgesia. PGE2 also triggers the development of mechanical hyperalgesia although its mechanisms are poorly understood. We hypothesized that PGE2 might also induce mechanical hyperalgesia through TRPV1 sensitization.

**Material & Methods:** All experiments were performed on CD-1 mice. Nociceptive pain to mechanical stimulus was evaluated by the paw pressure (450 g) test and nociceptive pain to heat stimulus was evaluated using the unilateral hot plate (55°C) test. PGE2- and GDNF-induced mechanical and thermal hyperalgesia were assessed by the same procedures as above, although at lower stimulation intensities (100 g and 42°C).

**Results:** The *in vivo* ablation of TRPV1-expressing neurons (by resiniferatoxin treatment) decreased nociceptive pain to heat stimulus without altering mechanical nociceptive pain. PGE2 administration induced both heat and mechanical hyperalgesia, and both types of sensory hypersensitivity were sensitive to resiniferatoxin treatment. The intraplantar administration of ruthenium red (a nonselective TRP antagonist), SB366791 (a selective TRPV1 antagonist), or H-89 (a PKA inhibitor) reversed PGE2-induced mechanical hyperalgesia. Any of these treatments modified GDNF-induced mechanical hyperalgesia, indicating that they do not induce nonselective actions affecting the sensitization of other neuronal populations different from TRPV1 neurons.

**Conclusions:** TRPV1 neurons are needed for nociceptive pain to heat stimulus but not for mechanical nociceptive pain. However, these neurons are involved in PGE2-induced hyperalgesia to both heat and mechanical stimuli, involving TRPV1 sensitization through the action of PKA.

**Acknowledgements:** MINECO (SAF2013-47481P and SAF2016-80540R), Junta de Andalucía (grant CTS 109), Laboratorios Esteve, FEDER funds and the Research Program of the University of Granada.

**Keywords:** TRPV1, prostaglandin E2, hyperalgesia

<footnote n="1">¹Department of Pharmacology, Biomedical Research Center and Institute of Neuroscience, University of Granada, Granada, Spain;</footnote>

<footnote n="2">²Department of Human Anatomy and Embryology, School of Medicine, University of Granada, Granada, Spain; ³Teófilo Hernando's Institute for Drug Discovery Medical School, Autonomous University of Madrid, Madrid, Spain</footnote>
P-155

ACTIVATION OF EXTRACELLULAR SIGNAL-REGULATED KINASES (ERK 1/2) IN THE LOCUS COERULEUS CONTRIBUTES TO PAIN-RELATED ANXIETY IN ARTHRITIC RATS

Borges G.<sup>1,2,3</sup>, Miguelez C.<sup>4</sup>, Neto F.<sup>2,3</sup>, Micó J.A.<sup>1,5,7</sup>, Ugedo L.<sup>4</sup>, Berrocoso E.<sup>5,6,7</sup>

<sup>1</sup>Neuropsychopharmacology and Psychobiology Research Group, Department of Neuroscience, University of Cádiz, 11003 Cádiz, Spain; <sup>2</sup>Departamento de Biomedicina-Unidade de Biologia Experimental, da Faculdade de Medicina da Universidade do Porto (FMUP), Porto, Portugal; <sup>3</sup>Instituto de Biologia Molecular e Celular (IBMC) e Instituto de Investigação e Inovação em Saúde (I3S), Universidade do Porto, Porto, Portugal; <sup>4</sup>Department of Pharmacology, Faculty of Medicine and Dentistry, University of the Basque Country UPV/EHU Leioa, Spain; <sup>5</sup>Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Instituto de Salud Carlos III, Madrid, Spain; <sup>6</sup>Neuropsychopharmacology and Psychobiology Research Group, Department of Psychology, University of Cádiz, 11510 Cádiz, Spain; <sup>7</sup>Instituto de Investigación e Innovación en Ciencias Biomédicas de Cádiz, INIBICA, Hospital Universitario Puerta del Mar, Cádiz, Spain

Chronic pain is now considered a disease with repercussions that reach far beyond hypersensitivity. Many studies have emphasized that chronic pain exponentially increases the risk of someone to suffer emotional disturbances characterized by persistent anxiety and low mood, creating a self-perpetuating cycle that magnifies and exacerbates the painful experience. However, the mechanisms underlying this co-morbidity are unknown. Prolonged arthritis can produce anxiety-like behavior in rats, along with enhanced phosphorylation of the extracellular signal-regulated kinase 1/2 (pERK1/2, a marker of activation and plasticity) in the Locus Coeruleus (LC). Therefore, we propose that ERK1/2 activation in the LC plays a crucial role in pain-related anxiety. To test this hypothesis, the monoarthritis model of painful arthritis was induced in rats by injection of complete Freund's adjuvant. The behavioral attributes of pain and anxiety, as well as LC function, were evaluated at an early (1 week, MA1W) and late-phase (4 weeks, MA4W) of the disease. Next, these procedures were repeated in MA4W rats in order to explore the effect of pERK1/2 blockade by intra-LC administration of the mitogen-activating extracellular kinase (MEK) inhibitor, SL327.

Results showed that pain was evident in monoarthritis rats after 1 week, although anxiety did not appear until 4 weeks later. This late-phase of the disease was accompanied by diminished tonic LC activity, which was coupled to exacerbated evoked LC responses to noxious stimulation of the inflamed and the healthy paw. When ERK1/2 activation was dampened by intra-LC administration of SL327, the exaggerated evoked response of MA4W rats was blocked. Importantly, SL327 did not change pain hypersensitivity but rather, it reversed both the anxiogenic-like behavior and the increase in pERK1/2 in the anterior cingulate cortex (ACC) of MA4W rats. Therefore, this study provides a direct evidence of how a subcortical structure interferes with ACC activity, leading to pain-related anxiety relief.

**Keywords:** anterior cingulate cortex; anxiety; Locus Coeruleus; pain

P-156

POSTOPERATIVE PAIN AND MORPHINE CONSUMPTION AFTER ULTRASOUND-GUIDED FEMORAL AND SCIATIC COMBINED NERVE BLOCK VS. NEUROSTIMULATION FOR FEMORAL AND SCIATIC COMBINED NERVE BLOCK OR NEUROSTIMULATION FOR FEMORAL NERVE BLOCK IN PRIMARY ELECTIVE TOTAL KNEE ARTHROPLASTY

Veronica P.M., Inmaculada B.E., Encarnación B.R., Aurelio G.L.

S of Anaesthesia, Virgen de la Victoria University Hospital, Malaga, Spain; D. Pharmacology and Clinical Therapeutic, Medicine School, Institute for Research in Biomedicine in Malaga (IBIMA), Spain; D. Pharmacology and Clinical Therapeutic, Medicine School, Institute for

Research in Biomedicine in Malaga (IBIMA). Spain; S of Anaesthesia. Virgen de la Victoria University Hospital. Malaga, Spain

Total knee arthroplasty injuries are extremely painful and merit prompt attention to adequate postoperative analgesia. We aim to compare femoral and sciatic ultrasound-guided combined nerve block vs. neurostimulation for femoral and sciatic combined nerve block or for femoral nerve block in postoperative pain in primary elective total knee prosthesis.

A three arms, prospective longitudinal study of patients having primary elective unilateral knee prosthesis and randomly assigned to catheter insertion guided by ultrasound or neurostimulation was done: (1) Ultrasound-guided femoral and sciatic combined nerve block (USFSCN) (N = 15); (2) Neurostimulation for femoral and sciatic combined nerve block (NSFSCN) (N = 17); (3) Neurostimulation for femoral nerve block (NSFN) (N = 11). Total analgesia (morphine) consumption after 48 hours was the primary endpoint. The postoperative pain intensity (visual analogue pain scale (VAS)) at post-anaesthetic recovery unit (PARU), 6, 24, 48 h, and during movement and postoperative complications were secondary outcomes.

43 patients (68.3 ± 8 years old, 77% female) subjected to elective unilateral knee prosthesis were enrolled. There were no differences in the demographic, anaesthetic and surgical variables between groups. Pain intensity was lower in the USFSCN group compared with NSFSCN and NSFN during the first 48 h post-surgery (% of intense pain at PARU/6 h/24 h/48 h); USFSCN 0.8/1.4/3.2/1.6; NSFSCN 5.6/8.3/7.5/3; NSFN 7.2/5.3/6.4/5.4. The average consumption of morphine within 48 h after surgery was similar in the groups USFSCN and NSFSCN (3 mg vs. 3.11 mg), and significantly lower than NSFN (4.19 mg) (*P* < 0.05). And the number of complications was significantly lower in the USFSCN group compared with NSFSCN and NSFN during the first 48 h of postoperative.

Ultrasound-guided femoral and sciatic combined nerve block presented better analgesia and was more safety than neurostimulation for femoral and sciatic combined nerve block or for femoral nerve block in primary elective total knee arthroplasty.

P-157

THE CHEMOKINE CCL4 INDUCES OPIOID ANALGESIA THROUGH LYMPHOCYTE STIMULATION AND INHIBITS INFLAMMATORY HYPERALGESIA IN MICE

García-Domínguez M., Lastra A., Vigil Martín C., R. Folgueras A., Cernuda-Cernuda R., Menéndez L., Baamonde A.

Área de Farmacología, Universidad de Oviedo, Oviedo, Spain

**Introduction:** Previous studies from our laboratory described that the s.c. administration of small doses of chemokine CCL4 (10–100 pg/Kg) to healthy mice produces analgesia through the release of endogenous opioids. The inhibition of this analgesia in mice treated with cyclophosphamide suggested the involvement of immune cells.

**Aims:** To elucidate the type of immune cells involved in the analgesic responses evoked by systemic CCL4 and to explore the ability of this chemokine to inhibit inflammatory hyperalgesia.

**Material and methods:** Thermal hyperalgesia was measured in Swiss CD1 mice by the unilateral hot plate test. Inflammation was induced by i.pl CFA one week before. The degree of immune cell depletion was checked with a differential hematology analyzer (Abacus). The expression of CCR5 in lymphocytes was assessed by immunofluorescence.

**Results:** The s.c. administration of CCL4 induced analgesia in mice treated with an antibody against granulocytes (antibody anti-Ly6G, 100 µg), but disappeared after selective lymphocytic depletion (antibody anti-CD4 30 µg + anti-CD8 20 µg). Supporting the involvement of these cells, the expression of CCR5, the main receptor for CCL4, was demonstrated in lymphocytes by immunofluorescence assays. The hyperalgesic response measured in CFA-inflamed mice was dose-dependently inhibited either after the systemic (0.1–1 pg/kg) or intrathecal (0.3–1 pg) administration of CCL4. However, whereas the
antihyperalgesic effect evoked by s.c. CCL4 was reversed by the CCR5 antagonist, DAPTA (3–10 mg/kg), or naloxone (0.1–1 mg/kg), only DAPTA (1–5 µg) but not naloxone (10 µg) inhibited its spinal antihyperalgesic effect.

**Conclusions:** Our results indicate that the systemic administration of CCL4 evokes opioid analgesia through the stimulation of CCR5 and the activation of lymphocytes. Furthermore, this chemokine counteracts inflammatory hyperalgesia evoked when administered either systemically or spinally, being the antihyperalgesic effect evoked after spinal administration unrelated to opioid mechanisms.
Fundings came from FICYT (FC-15-GRUPIN14-125).

**Keywords:** CCL4, CCR5, DAPTA, Inflammation, Naloxone

P-158

TREATMENT OF NEUROPATHIC PAIN ASSOCIATED TO
THE SPARED NERVE INJURY MODEL IN MICE: ROLE OF
SIGMA-1 RECEPTORS

Bravo-Caparrós I.¹,², Nieto F.R.¹,², Perazzoli G.³, Baeyens J.M.¹,²,
Cobos E.J.¹,²

¹Department of Pharmacology, School of Medicine, University of Granada, Granada, Spain; ²Institute of Neuroscience, Biomedical Research Center, University of Granada, Armilla, Granada, Spain;

³Department of Human Anatomy and Embryology, School of Medicine, University of Granada, Granada, Spain

**Background and aims:** Sigma-1 receptors (σ₁Rs) are an emerging target for the treatment of neuropathic pain. The role of σ₁Rs in neuropathic pain induced by nerve compression is known, but their participation on peripheral neuropathy induced by nerve transection is unexplored. Therefore, we investigated the effect of pharmacological and genetic blockade of σ₁Rs in the neuropathic pain associated to the sciatic nerve spared nerve injury (SNI) model. This model involves a lesion of two of the three terminal branches of the sciatic nerve, leaving the sural nerve intact.

**Methods:** SNI was performed on female CD1 wild-type (WT) and σ₁Rs knockout (σ₁Rs-KO) mice. Mechanical allodynia (von Frey test), cold-allodynia (acetone test) and heat hyperalgesia (Hargreave's test) were tested previously to SNI and during three weeks following injury. The effect of acute σ₁Rs antagonist S1RA (16–64 mg/kg, s.c.) and agonist PRE-084 (32 mg/kg, s.c.) were tested seven days after SNI. Animals were also treated with S1RA (25 mg/kg i.p.) twice daily for 10 days.

**Results:** WT mice developed prominent cold and mechanical allodynia after SNI, whereas σ₁Rs-KO did not develop cold allodynia and showed reduced mechanical allodynia. These effects were mimicked by the administration of S1RA to neuropathic WT mice, and the effects of the σ₁ antagonist were reversed by PRE-084. σ₁Rs-KO and WT mice showed a similar thermal hyperalgesia after SNI. However, S1RA abolished SNI-induced thermal hyperalgesia in WT mice, an effect which was reversed by PRE-084. Mechanical allodynia and cold allodynia were reduced by the repeated administration of S1RA.

**Conclusion:** σ₁ receptors have a relevant role in SNI-induced neuropathic pain. The pharmacological antagonism of this receptor can be useful to prevent and/or treat neuropathic pain produced by nerve section.

**Keywords:** Neuropathic pain; Allodynia; hyperalgesia; Sigma-1 receptors; S1RA

**Acknowledgments:** Spanish Government (SAF2016-80540-R, FPU, Juan-de-la-Cierva-Incorporación-grants), Junta de Andalucía (P11-CTS-7649) and the Research Program of University-of-Granada.

P-159

DEVELOPMENT OF PHENOTYPIC IN VITRO MODELS OF
PAIN IN EARLY DRUG DISCOVERY

Martínez A.L.¹, Monroy X.², Brea J.¹, Merlós M.², Burgueño J.², Loza M.I.¹

¹Center of Research on Molecular Medicine and Chronic Diseases (CIMUS). Universidade de Santiago de Compostela, Santiago de Compostela (A Coruña); ²Laboratorios Esteve, Parc Científic de Barcelona, Barcelona

**Background:** Despite considerable efforts for treating neuropathic pain, new analgesics identified in preclinical studies failed in clinical trials. One explanation could be that the sole modulation of a given target may not be enough to have a therapeutic effect. Another explanation is the scarce translationality of *in vitro* assays in early drug discovery, where immortalized cells unrelated to the disease and overexpressing the target of interest are used.

**Aim of the Study:** Our aim is to develop High-Throughput Screening (HTS) phenotypic and translational assays to discover new analgesics for neuropathic pain using immortalized Dorsal Root Ganglion (DRG) neuronal cells better resembling the physiology of nociceptors than conventional cell cultures, while keeping throughput, assay sensitivity and reproducibility for HTS.

**Methods:** We evaluated several protocols to differentiate immortalized DRG cell lines (F11 and ND7/23) to a neuronal phenotype considering both their morphology and expression of neuronal markers. Also, we functionally evaluated their response to activation by ATP and to the depolarization induced by KCl using dynamic mass redistribution label-free measurements (Epic system, Perkin-Elmer).

**Results:** We found an effective protocol to differentiate both cell lines to DRG-like neurons using 30 µM forskolin, 0.5 mM dibutyryl cAMP and 0.5% FBS or 60 µM forskolin, 1 mM dibutyryl cAMP, 1,3 ng/ml NGF and 0.5% FBS for F11 and ND7/23 respectively, as shown by the advent of neuron-like processes and the increased expression of TrkA receptor. Both cell lines responded to ATP and KCl in a concentration-dependent manner in the label-free assay, obtaining with ATP an EC₅₀ of 4·10⁻³M and 7·10⁻³M in F11 and ND7/23 cells, respectively. In the case of KCl, F11 cells responded with an EC₅₀ of 0.2M.

**Conclusions:** These differentiated cell lines could represent new biological tools for establishing novel phenotypic *in vitro* models for HTS of drugs to treat neuropathic pain.

**Keywords:** High Throughput Screening (HTS); *in vitro* assay; neuropathic pain; Dynamic Mass Redistribution (DMR)

P-160

TREATMENT WITH SULFORAPHANE ENHANCES THE
ANTINOCICEPTIVE EFFECTS OF A OPIOID RECEPTORS IN
DIABETIC MICE: MECHANISMS IMPLICATED

McDonnell C.¹,², Leánez S.¹,², Pol O.¹,²

¹Grup de Neuroarmacologia Molecular, Institut d'Investigació
Biomèdica Sant Pau, Barcelona, Spain; ²Institut de Neurociències,
Universitat Autònoma de Barcelona, Barcelona, Spain

**Background:** The involvement of heme oxygenase 1 (HO-1) in the modulation of the antinociceptive effects of opioids in type 1 diabetes has been demonstrated but the role played by the transcription factor Nrf2 in the regulation of painful neuropathy and in the effects and expression of δ-opioid receptors (DOR) in type 2 diabetic mice have not studied.

**Methods:** In male BKS.Cg-m+/+Leprdb/J (db/db) mice, the anti-allo-dynic effects produced by the administration of a Nrf2 transcription factor activator sulforaphane (SFN) alone and combined with two DOR agonists, [d-Pen(2),d-Pen(5)]-Enkephalin (DPDPE) and (+)-4-[(αR)-α-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N diethylbenzamide (SNC-80) were evaluated. The effects of SFN on glucose levels and body weight as well as on the proteins levels of Nrf2, HO-1, NAD(P)H: quinone oxidoreductase 1 (NQO1),
MAPKs (JNK) and DOR in sciatic nerve from db/db mice were also assessed.

**Results:** This study showed that the administration of SFN dose dependently reversed mechanical allodynia, reduced hyperglycemia and body weight gain associated to type 2 diabetes and significantly increased the anti-allodynic effects of DPDPE and SNC-80 in db/db mice. This treatment also enhanced the protein levels of HO-1, normalized the down regulation of Nrf2, NQO1 and DOR, and inhibited JNK phosphorylation in the sciatic nerve of diabetic mice. Our data indicated that SFN treatment is effective in reversing mechanical allodynia and enhancing DOR antinociceptive effects by activating the Nrf2/HO-1/NQO1 signaling pathway, decreasing the phosphorylation of JNK and normalizing DOR expression in db/db mice.

**Conclusions:** These results propose SFN, alone and combined with DOR agonists, as interesting approaches for the treatment of painful diabetic neuropathy associated to type 2 diabetes in mice. This work was supported by Ministerio de Economía y Competitividad, Instituto de Salud Carlos III and Fondo Europeo de Desarrollo Regional (FEDER), Unión Europea [Grant PI1400927].

# P-161
## EFFECTS OF VASCULAR ENDOTHELIAL GROWTH FACTOR-A₁₆₅B AND SRPK1 INHIBITION IN THE MONOSODIUM IODOACETATE MODEL OF OSTEOARTHRITIS IN RAT

Almahasneh F.¹,², Beazley-Long N.¹,², Blackley Z.¹, Bates D.O.³, Donaldson L.¹,²

¹School of Life Sciences, University of Nottingham, UK; ²Arthritis Research UK Pain Centre, University of Nottingham, UK; ³Cancer Biology, School of Medicine, University of Nottingham, UK

Osteoarthritis (OA) is a major cause of musculoskeletal pain worldwide, and it is still inadequately managed. Serine-arginine protein kinase 1 (SRPK1) controls the alternative splicing of vascular endothelial growth factor-A into the pro-nociceptive VEGF-A₁₆₅a variant and the anti-nociceptive VEGF-A₁₆₅b. This study evaluated the effects of VEGF-A₁₆₅b and SRPK1 inhibition on pain behaviour and chondropathy severity in the monosodium iodoacetate (MIA) model of OA in rats.

Male Wistar rats (n = 56, 200-250 g) were used. In study 1, arthritis was induced with intra-knee-joint injection of MIA (1 mg/50 µl saline, n = 23). Controls were untreated (n = 9). Rats were treated with: VEGF-A₁₆₅b (20 ng/g body weight, days 0-14) followed by PBS (days 15-28, twice weekly, I.P.), or PBS (d0-14) followed by VEGF-A₁₆₅b (d15-28). In study 2, MIA rats (n = 16) were treated with SRPK1 inhibitor EXN109 (0.8 µg/g body weight in 1% DMSO). Weight bearing and mechanical withdrawal thresholds were measured twice weekly. Rats were euthanized (100 mg pentobarbital i.p.) on day 28, and knee joint macro- and microscopic chondropathy assessed.

Weight bearing asymmetry was significantly reduced in MIA/VEGF (d0-14) rats (weight borne ipsilaterally 44.7 ± 2.7% body weight) compared to controls (38.6 ± 2.7%) on days 25 and 28. EXN109 had no significant effect on pain behaviour (weight borne ipsilaterally 44.4 ± 1.3% body weight) compared to controls (42.7 ± 1.2%) (two-way ANOVA, post-hoc Tukey's tests).

Both gross and microscopic chondropathy scores were significantly higher in MIA animals (gross score: 15.2 ± 2.0; microscopic score 12.2 ± 0.9) and in all treated animals (VEGF₁₆₅b(d0-14) gross: 11.2 ± 1.3; microscopic 10.7 ± 1.0, EXN109 gross: 14.3 ± 0.9; microscopic: 8.2 ± 1.3) than in controls (gross: 0.2 ± 0.1; microscopic: 1.7 ± 0.4, one-way ANOVA).

Systemic VEGF-A₁₆₅b has an anti-nociceptive action, but no effect on joint damage in the MIA model of OA in rat only when given early in disease development. The lack of effect of EXN109 on OA pain and chondropathy is probably due to the early stage of drug development.

**Keywords:** osteoarthritis, pain, VEGF, SRPK

# P-162
## PLASMA LEVELS OF THERAPEUTIC DRUGS SIGNIFICANTLY INHIBIT BLOOD-BRAIN BARRIER ENDOTHELIAL CELL ABC TRANSPORTERS

Shubbar M.H., Penny J.I.

Division of Pharmacy & Optometry, School of Health Sciences, Stopford Building, University of Manchester, Manchester M13 9PT, UK

**Introduction:** Clinically relevant interactions between therapeutic drugs and transporters, including ATP-binding cassette (ABC) efflux transporters, are of growing interest, as highlighted by the FDA [1]. Inhibition or induction of ABC transporters can cause pharmacokinetic interactions leading to unpredicted toxicities or sub-optimal treatment. ABCB1 and ABCG2 are highly expressed in blood-brain barrier endothelial cells, and modulation of their activities may influence CNS drug penetration of ABCB1 and ABCG2 substrates [2].

**Methods:** Primary porcine cerebromicrovascular endothelial cells were isolated and maintained as described previously [3]. The effect of indomethacin, olanzapine, chlorpromazine and glibenclamide, at concentrations reported in plasma (5 µM indomethacin, 650 nM olanzapine, 1 µM chlorpromazine, 1 µM glibenclamide), on cell viability was measured using the Neutral Red assay. The impact of drugs on ABCB1 and ABCG2 transporter activity was determined by measuring intracellular calcein and Hoechst 33342 accumulation, respectively, in cells pre-treated with these drugs.

**Results:** The drugs demonstrated no significant effect on the viability of porcine cerebromicrovascular endothelial cells at the concentrations used.

Indomethacin, olanzapine, chlorpromazine and glibenclamide all significantly increased intracellular accumulation of calcein, indicating inhibition of ABCB1. Whereas indomethacin, olanzapine, chlorpromazine significantly increased intracellular accumulation of Hoechst 33342, indicating inhibition of ABCG2.

**Conclusion:** This is the first study to demonstrate plasma levels of therapeutic drugs significantly inhibit ABCB1 and ABCG2 in primary porcine cerebromicrovascular endothelial cells. The findings suggest that these drugs, which are widely used in polypharmacy, have the potential to influence the CNS permeability of co-administered ABCB1 and ABCG2 drug substrates.

### References:

1. Huang, S.M., et al., *Drug interaction studies: study design, data analysis, and implications for dosing and labeling*. Clinical Pharmacology & Therapeutics, 2007. **81**(2): p. 298-304.

2. Marchetti, S., et al., *Concise review: Clinical relevance of drug-drug and herb-drug interactions mediated by the ABC transporter ABCB1 (MDR1, P-glycoprotein)*, in *Oncologist* 2007. p. 927-941.

3. Cantrill, C.A., et al., *An immortalised astrocyte cell line maintains the in vivo phenotype of a primary porcine in vitro blood-brain barrier model*. Brain research, 2012. **1479**: p. 17-30.

# P-163
## IMPACT OF CEREBRAL ISCHEMIA/REPERFUSION AND URIC ACID TREATMENT ON MIDDLE CEREBRAL ARTERY ANGIOGENESIS IN NORMOTENSIVE AND SPONTANEOUSLY HYPERTENSIVE RATS

Masip N., Lapaz E., Jiménez-Altayó F., Vila E.

Departament de Farmacologia, de Terapèutica i de Toxicologia, Institut de Neurociències, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain

High blood pressure is the single most important risk factor for stroke and hypertensive patients have poor post-stroke functional outcomes. Angiogenesis plays a role in stroke recovery, though excessive neovascularization may be detrimental. It is unclear whether hypertension may alter angiogenesis after stroke and the effect of uric acid (UA) antioxidant therapy, which is showing encouraging neuroprotective
effects in clinical trials. We analysed the influence of cerebral ischemia (90 min)/reperfusion (24 h) on the time course of *in vitro* middle cerebral artery (MCA) angiogenic growth in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) treated (i.v.; 120 min after MCA occlusion) with UA (16 mg/kg) or vehicle (Locke's buffer). Ipsilateral and contralateral to ischemia MCA explants were cultured in Matrigel and vessel sprouting was measured from day 4 through day 14. Systolic blood pressure (P < 0.001) and infarct volumes(P = 0.05) were larger in SHR than in WKY, and UA attenuated (P < 0.05) infarct volume in both strains. Ischemia/reperfusion induced an early (from day 4 to 7) increase in angiogenesis in ipsilateral MCA from WKY (P < 0.05) and SHR (P < 0.01) compared to sham-operated rats that was prevented by UA only in SHR. Vessel sprouting was higher (P < 0.05) at day 4 (230.3 ± 16.4 µm, n = 6) and lower (P < 0.05) at day 14 (482.4 ± 34.8 µm, n = 6) in SHR than in WKY (day 4: 139.2 ± 34.9 µm, n = 5; day 14: 591.0 ± 46.8 µm, n = 5) after ischemia/reperfusion, an effect that was prevented by UA. In conclusion, transient cerebral ischemia potentiates early stage angiogenesis in the ipsilateral MCA independently of high blood pressure. However, hypertension is linked to early exacerbated and late impaired angiogenic growth after ischemia/reperfusion. UA treatment does not modify early increased angiogenesis in WKY, while it prevents both early and long-term alterations in SHR.

**Supported by Ministerio de Ciencia e Innovación (SAF2014-56111-R):**

**Keywords:** cerebral ischemia/reperfusion; hypertension; angiogenesis; antioxidant treatment

P-164

SIMVASTATIN BLOCKS SOLUBLE SSAO/VAP-1 RELEASE IN EXPERIMENTAL MODELS OF CEREBRAL ISCHEMIA: POSSIBLE BENEFITS FOR STROKE-INDUCED INFLAMMATION CONTROL

Sun P.¹, Hernandez-Guillamón M.², Campos-Martorell M.², Simats A.², Montaner J.², Unzeta M.¹, Solé M.¹

¹Departament de Bioquímica i Biologia Molecular. Institut de Neurociències. Universitat Autònoma de Barcelona, Bellaterra (Barcelona), Spain; ²Neurovascular Research Laboratory, Institut de Recerca Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain

Beyond cholesterol reduction, statins mediate their beneficial effects on stroke through pleiotropic actions. They have shown anti-inflammatory properties by different mechanisms, including the inhibition of the NF-κB transcriptional activity and the consequent increase and release of adhesion molecules. We have studied simvastatin effects on the vascular enzyme semicarbazide-sensitive amine oxidase/vascular adhesion protein 1 (SSAO/VAP-1), which is involved in the stroke-mediated brain injury. SSAO/VAP-1 has leukocyte-binding capacity and mediates the expression of other adhesion proteins through signaling molecules generated from its catalytic activity, which when overproduced induce vascular damage. Our results indicate that soluble SSAO/VAP-1 is released to the bloodstream after an ischemic stimulus, in parallel with an increase in E-selectin and VCAM-1 in the contralateral brain side. Simvastatin blocks the soluble SSAO/VAP-1 release and prevents E-selectin and VCAM-1 overexpression as well. Thus, simvastatin efficiently blocks SSAO/VAP-1-mediated leukocyte adhesion, although it is not an enzymatic inhibitor of SSAO. In addition, simvastatin-induced changes in adhesion molecules are greater in human brain endothelial cell cultures expressing SSAO/VAP-1, compared to those non-expressing it, indicating some synergic effect with SSAO/VAP-1. We think that combined therapy with SSAO/VAP-1 inhibitors and statins could enhance the beneficial effects of both individual treatments in stroke patients.

**Keywords:** stroke; statins; SSAO/VAP-1; inflammation

P-165

OLIVE LEAF EXTRACT EXHIBITS IMMUNOMODULATORY ACTIVITY IN HUMAN PBMCS AND ANTI-INFLAMMATORY EFFECT IN THE DSS MODEL OF MOUSE COLITIS

Vezza T.¹, Algieri F.¹, Rodríguez-Nogales A.¹, Garrido-Mesa J.¹, Utrilla M.P.¹, Talhaoui N.²,³, Gómez-Caravaca A.M.²,³, Segura-Carretero A.²,³, Rodríguez-Cabezas M.E.¹, Gálvez J.¹

¹Department of Pharmacology, CIBER-EHD, ibs.GRANADA, CIBM, University of Granada, Spain; ²Department of Analytical Chemistry, University of Granada.Avda. Fuentenueva s/n, 18071 Granada, Spain;

³Research and Development of Functional Food Centre (CIDAF), PTS Granada, Avda.delConocimiento s/n., EdificioBioregión, Granada, Spain; (Department of Pharmacology, CIBER-EHD, ibs.GRANADA, CIBM, University of Granada, Granada 18009, Spain)

**Introduction:** Olive (Oleaeuropea) leaf extract (OLE) is used in Mediterranean traditional medicine as an anti-inflammatory remedy, most probably due to its presence of antioxidant phenolic compounds, like oleuropeoside. It would be interesting to validate it use for the treatment of inflammatory conditions associated with oxidative stress in humans, like inflammatory bowel disease.

**Aim:** To evaluate the intestinal anti-inflammatory properties of an olive leaf extract in the dextran sodium sulfate (DSS) model of mouse colitis, which resembles human IBD.

**Methods:** Male C57BL/6J mice were assigned into five groups: non-colitic, colitic control and colitic treated groups with olive leaf extract (0.5-1-10 mg/kg).Colitis was induced by incorporating DSS(3%) in the drinking water for 5 days. Treatment started the same day of colitis induction, and maintained until one week after the establishment of the colitic process. The inflammatory status was evaluated macroscopically, assigning a disease activity index (DAI), and biochemically by determining the colonic expression of mediators involved in the inflammatory response or in the intestinal epithelial barrier function. In addition, *in vitro* immunomodulatory properties of the extract were determined when incubated (0.1–100 µg/ml) with peripheral blood mononuclear cells(PBMCs)obtained from healthy and Crohn's disease patients.

**Results:** The treatment with the extract improved the recovery of the colitic mice, since a significant reduction in DAI values was observed. This effect was associated with a reduced expression of colonic pro-inflammatory mediators(IL-1β, IL-6, TNFα, ICAM-1 and MIP-2),while increasing that of key players of the intestinal epithelial integrity(occludin, ZO-1 and MUC-3). Besides, it displayed immunomodulatory properties *in vitro* since it decreased pro-inflammatory cytokines production in LPS-stimulated PBMCs.

**Conclusion:** OLE showed intestinal anti-inflammatory activity in the DSS model of mouse colitis, maybe related to its antioxidant properties, which may result in the downregulation of the immune response and the improvement of the intestinal epithelial barrier function. In addition, this extract has a direct effect on immune cells, as demonstrated in the *in vitro* studies.

**Keywords:** Inflammatory bowel disease, mouse experimental colitis, olive leaf extract, polyphenols

P-166

NEW CURCUMIN-COUMARIN DERIVATIVES AND RESVERATROL REDUCE PATHOPHYSIOLOGICAL PROCESSES ASSOCIATED WITH PANCREATITIS AND PANCREATIC CANCER

Bugallo A., Luaces-Regueira M.*, Campos-Toimil M.*

Pharmacology of Chronic Diseases (CD Pharma), Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Santiago de Compostela, Spain. *Equal contribution.

**Background:** Pancreatic diseases have not a curative treatment nowadays. Progressive inflammatory unbalance and physiopathology
associated events, as intracellular trypsin activity and abnormal
increase of the cytoplasmic concentration of Ca²⁺ ([Ca²⁺]ᵢ), are com-
mon pathways for both pancreatitis and pancreatic cancer.

**Methods:** In the present study, we have used an *in vitro* model, based on the stimulation of the pancreatic acinar cells (PAC) with a supra-maximal concentration (100 nM) of the cholecystokinin hormone (CCK) to evaluate the activity of two new hybrid curcumin-coumarin derivatives (H1K3 and H1K5) and resveratrol against several physiological processes associated with both pancreatic diseases, such as premature trypsin intracellular activation, abnormal [Ca²⁺]ᵢ or PAC necrosis. All the measures were carried out with fluorescence quantitative techniques with different specific fluorochromes (rhodamine, fluo4 and propidium iodide).

**Results:** Our experiments show that H1K3 and H1K5 (10, 100 µM) and resveratrol (200 nM, but not 20 nM) significantly inhibit both CCK-induced intracellular trypsin activity (at min 20 and 40) and PAC necrosis. Also, H1K3 and H1K5 (10, 100 µM) significantly reduce CCK-induced [Ca²⁺]ᵢ increases in PAC.

**Conclusions:** Our results with H1K3 and H1K5 confirm the potential therapeutic interest of the combination of curcumin-coumarin structures on the cellular basis of pancreatitis and pancreatic cancer. In addition, a potential beneficial effect of resveratrol against both pancreatic pathologies is confirmed. Further studies relating those findings with inflammatory process as main target of these new molecules will be performed.

**Keywords:** pancreatitis, pancreatic cancer, curcumin-coumarin, resveratrol