Role of ABC-Transporters in Epileptogenesis and Pharmacoresistant Epilepsy

According to a recently published article by our group [The complexity of roles of P-glycoprotein in refractory epilepsy: pharmacoresistance, epileptogenesis, SUDEP and relapsing marker after surgical treatment ADMET & DMPK 3(2) (2015) 110-121], we have written a chapter related to these concepts

Seizures Induces Hypoxia and Hypoxia Induces Seizures: A Perverse Relationship that Increases the Risk of SUDEP

He uttered a cry and was seen to be rubbing his hands together. His pulse wasimmediately examined for but was not palpable.Hypoxia is a biological stimulus capable of promoting both rescue and survivalmechanisms, as well as triggering a sequence of irreversible events that lead to deathof cell, tissue and even individual. Brain and heart functions depend critically onadequate energy supply and they are highly susceptible to hypoxic conditions.Adequate oxygen supply is needed for the brain and heart to metabolize glucose asits major energy source. But how many type of hypoxia we know, and how muchsevere is the hypoxic condition, when more than one type of hypoxia aresimultaneously acting. Four types of hypoxia are distinguished in medicine: α- thehypoxemic hypoxia, due to a decrease in the amount of breathable oxygen orcardiopulmonary failure, b- the anemic hypoxia related with decreased amount offunctional hemoglobin, c- the stagnant hypoxia, secondary to reduced or unevenlydistributed flow of blood distribution to the tissues, and mainly result from heartdisease that impairs the circulation; and d- the histotoxic hypoxia, in which the tissuecells are poisoned and are therefore unable to make proper use of oxygen.Fil: Auzmendi, Jerónimo Andrés. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Lazarowski, Alberto Jorge. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Bioquímica Clínica; Argentin

Convulsive Stress Mimics Brain Hypoxia and Promotes the P-Glycoprotein (P-gp) and Erythropoietin Receptor Overexpression. Recombinant Human Erythropoietin Effect on P-gp Activity

Erythropoietin (EPO) is not only a hormone that promotes erythropoiesis but also has a neuroprotective effect on neurons attributed to its known anti-apoptotic action. Previously, our group has demonstrated that recombinant-human EPO (rHu-EPO) can protect neurons and recovery motor activity in a chemical focal brain hypoxia model (Merelli et al., 2011). We and others also have reported that repetitive seizures can mimic a hypoxic- like condition by HIF-1α nuclear translocation and high neuronal expression P-gp. Here, we report that a single 20-min status epilepticus (SE) induces P-gp and EPO-R expression in cortical pyramidal neurons and only P-gp expression in astrocytes. In vitro, excitotoxic stress (300 μM glutamate, 5 min), can also induce the expression of EPO-R and P-gp simultaneously with both HIF-1α and NFkB nuclear translocation in primary cortical neurons. Primary astrocytes exposed to chemical hypoxia with CoCl2 (0.3 mM, 6 h) increased P-gp expression as well as an increased efflux of Rhodamine 123 (Rho123) that is a P-gp substrate. Tariquidar, a specific 3er generation P-gp-blocker was used as an efflux inhibitor control. Astrocytes treated with rHu-EPO showed a significant recovery of the Rho123 retention in a similar way as seen by Tariquidar, demonstrating for first time that rHu-EPO can inhibit the P-gp-dependent efflux activity. Taking together, these data suggest that stimulation of EPO depending signaling system could not only play a central role in brain cell protection, but this system could be a new tool for reverse the pharmacoresistant phenotype in refractory epilepsy as well as in other pharmacoresistant hypoxic brain diseases expressing P-gp.Fil: Merelli, Amalia Margarita. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Fisiopatología y Bioquímica Clínica; ArgentinaFil: Ramos, Alberto Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Lazarowski, Alberto Jorge. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Fisiopatología y Bioquímica Clínica; ArgentinaFil: Auzmendi, Jerónimo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentin

The complexity of roles of P-glycoprotein in refractory epilepsy: Pharmacoresistance, epileptogenesis, SUDEP and relapsing marker after surgical treatment

As described initially from clinical and experimental studies, P-glycoprotein (P-gp) plays a central role in the pharmacoresistance of epilepsy, acting by efflux of AEDs mainly at blood brain barrier (BBB) level. However, repetitive seizures can produce both brain and heart P-gp overexpression. Because P-gp activity induces membrane depolarization, its neuronal expression could be acting in the intrinsic mechanism of epileptogenesis, and its heart expression, can be a high risk factor of death, after severe-continuo convulsive stresses as in fatal status epilepticus or in SUDEP. Additionally, because P-gp is also a stem cell marker, we suggests that its constitutive overexpression in dysplastic neurons from brain epileptogenic areas observed in patients with refractory epilepsies, should be addressed as a risk factor of seizures relapse after surgical treatment. Here we discuss these concepts, based on our own clinical and experimental experiences, and reviewing the current literature on these subjects

Is cannabidiol a drug acting on unconventional targets to control drug-resistant epilepsy

Cannabis has been considered as a therapeutic strategy to control intractable epilepsy. Several cannabis components, especially cannabidiol (CBD), induce antiseizure effects. However, additional information is necessary to identify the types of epilepsies that can be controlled by these components and the mechanisms involved in these effects. This review presents a summary of the discussion carried out during the 2nd Latin American Workshop on Neurobiology of Epilepsy entitled “Cannabinoid and epilepsy: myths and realities.” This event was carried out during the 10th Latin American Epilepsy Congress in San José de Costa Rica (September 28, 2018). The review focuses to discuss the use of CBD as a new therapeutic strategy to control drug-resistant epilepsy. It also indicates the necessity to consider the evaluation of unconventional targets such as P-glycoprotein, to explain the effects of CBD in drug-resistant epilepsy.Fil: Rocha, Luisa. CENTRO DE INVESTIGACIÓN Y DE ESTUDIOS AVANZADOS ; INSTITUTO POLITÉCNICO NACIONAL;Fil: Lizette Frías Soria , Christian. CENTRO DE INVESTIGACIÓN Y DE ESTUDIOS AVANZADOS ; INSTITUTO POLITÉCNICO NACIONAL;Fil: Ortiz, José G.. Universidad de Puerto Rico; Puerto RicoFil: Auzmendi, Jerónimo Andrés. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Fisiopatología y Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Lazarowski, Alberto. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Fisiopatología y Bioquímica Clínica; Argentin

Tumor associated fibroblast: impact on osteosarcoma primary and metastatic tumoral microenvironment and treatment response

Tumor associated fibroblast (TAF) have been implicated in almost every aspect of tumoral biology. Of relevance TAF could be modulating response to treatment and overall microenvironment development. Given that Osteosarcoma (OS) have the same 5-year survival rate for metastatic and treatment resistant patients since 1970 ́s, we dediced to investigate the role of TAF in OS primary and metastatic niches. Aim: Evaluate TAF and human OS cell lines interaction in primary and pulmonary metastatic environments. To analyze if metastatic OS cell line has a higher inducing power than non-metastatic OS cell line analyzing the expression of different ABC transporters im-plicated in chemoresistance and the ability to exclude doxorubicin and rhodamine. Methods: The expression of ABC transporters was analyzed by RT-qPCR on conditioned fibroblast. Rhodamine 123 exclution assay was used to determine the activity of P-glycoprotein (P-gp) mediated transport and doxorubicin (DOX) exclution was performed to analysis the overall ABC-related chemoresistant capacity. To evaluate the interaction of fibroblast with metastatic (LM7) and non-metastatic (SAOS2) OS human cells hetero – spheroid formation assays were performed. Results: LM7 conditioned medium (CM) induced an overall upregulation of ABC transporters in comparison with SAOS2 CM. Conditioned fibroblast with LM7 CM showed lower levels of intracellular DOX and Rhodomine in comparison with SAOS2 CM fibroblast. Mixed spheroid compose of fibroblast ans OS cell lines display a lower area and more compact than single type aggregates. OS has not changed the 5-year rate survival for metastatic patients since the 70’, so the need to understand aspects of OS metastatic biology and chemoresistance could be helpful to develop new treat-ments to this group. Knowing aspects of the associated stroma and in particular TAF, could allow the development of new therapeutic possibilities targeting the tumoral associated stroma.Fil: Valenzuela Alvarez, Matias Juan Pablo. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; ArgentinaFil: Rizzo, Matias Eduardo. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; ArgentinaFil: Auzmendi, Jeronimo. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Lazarowski, Alberto. Universidad de Buenos Aires; ArgentinaFil: Bolontrade, Marcela Fabiana. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; ArgentinaLXVI Reunión Anual de la Sociedad Argentina de Investigación Clínica: LXIX Reunión Anual de la Sociedad Argentina de Inmunología; LIII Reunión Anual de la Asociación Argentina de Farmacología Experimental y XI Reunión Anual de la Asociación Argentina de NanomedicinasArgentinaSociedad Argentina de Investigación ClínicaSociedad Argentina de InmunologíaAsociación Argentina de Farmacología Experimenta

Iron: from normal homeostasis to death by ferroptosis

El hierro (Fe) es un elemento vital para casi todos los organismos debidoa su facilidad para donar y aceptar electrones. Es cofactor de muchasproteínas y enzimas necesarias para la adecuada utilización del oxígeno yla generación de energía. Su desregulación se relaciona a procesos de estrés oxidativo y muerte celular mediada por Fe(II) denominada ferroptosis.Las células de mamíferos utilizan múltiples mecanismos para garantizarla adquisición del hierro como nutriente esencial, que se encuentra oxidado [Fe(III)], y que debe ser reducido a Fe(II) para su adecuada utilización intracelular. Cada etapa de transferencia del hierro a través de lasmembranas biológicas exige una reconversión de su estado de oxidadoa reducido y viceversa, dependiendo del paso metabólico implicado. Ladistorsión de dichos procesos se asocia con varias enfermedades: desdela deficiencia de hierro debida a defectos en la adquisición o distribucióndel metal, que causa anemia, a la sobrecarga de hierro que resulta de unaabsorción excesiva de hierro o en una utilización defectuosa, que causauna sobreoferta de Fe(II) en los tejidos y que lleva a un daño oxidativoy a la muerte celular. Existen múltiples mecanismos regulatorios que enconjunto aseguran el equilibrio en la homeostasis del hierro. Esta actualización describe los avances recientes en las vías reguladoras del hierro, así como en los mecanismos subyacentes al tráfico de dicho elemento desde su absorción, principalmente biodistribución y su uso intracelular, quizás el área más importante donde se define su adecuada utilización o la muerte celular por ferroptosis.Iron (Fe) is a vital element for almost all organisms due to its ability to donate and accept electrons with relative ease. It serves as a cofactor for many proteins and enzymes necessary for the proper use of oxygen and energy generation, and its deregulation is related to the processes of oxidative stress and iron-mediated cell death called ferroptosis. Mammalian cells use multiple mechanisms to ensure the acquisition of iron as an essential nutrient, which is normally oxidised in the form of Fe(III) and must be reduced to Fe(II) for adequate intracellular use. Each stage of iron transfer across biological membranes requires a reconversion of its state from oxidised to reduced and vice versa, depending on the metabolic step involved. Distortion of these processes is associated with various diseases, such as iron deficiency due to defects in the acquisition or distribution of the metal that causes anemia, as well as iron overload from excessive iron absorption or defective use, which results in an oversupply of Fe(II) in tissues leading to oxidative damage and cell death. There are multiple regulatory mechanisms that together ensure the balance in iron homeostasis. This update describes the recent advances in the iron regulatory pathways, as well as in the mechanisms underlying iron trafficking from its absorption, mainly biodistribution and its intracellular use, perhaps the most important area where its adequate utilisation or cell death by ferroptosis is defined.Fil: Lazarowski, Alberto Jorge. No especifíca;Fil: Vitale, Arturo Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Auzmendi, Jerónimo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pomilio, Alicia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Progressive heart P-glycoprotein (P-gp) overexpression after experimental repetitive seizures (ERS) associated with fatal status epilepticus (FSE): Is it related with SUDEP?

Patients with refractory epilepsy (RE) have increased risk of Sudden Unexpected Death in Epilepsy (SUDEP), where acute and fatal heart failure is suspected. High seizure frequency, polypharmacy, changes in dosing, persistent low AED levels or poor adherence to therapy are the greatest risk factors of SUDEP and are also features observed in RE. We evaluated the progressive P-gp overexpression in heart, related with the development of fatal status epilepticus (FSE) after experimental repetitive seizures (ERS). Male Wistar rats (180–230g) were daily injected (i.p) with Pentylenetetrazole (PTZ; 45mg/kg; n=18) or saline (Controls; n=6). Severity of seizures was recorded. Four PTZ-treated rats were sacrificed at 4th-7th day respectively. Ten remaining rats, underwent same treatment until develop fatal status epilepticus (FSE). Brains and hearts were studied by immunofluorecent method for P-glycoptrotein (P-gp) expression. Seizures were observed each day of PTZ treatment, associated with a progressive P-gp overexpression in heart and FSE at 9th day. Using the same PTZ model, we previously demonstrated that progressive brain P-gp overexpression contributes to cell membrane depolarization of hippocampus and neocortex. These are the first evidences showing that ERS induces a simultaneous and progressive P-gp overexpression in brain and heart associated with FSE, and suggests a role for this pattern expression of P-gp as risk factor for death during SE. The simultaneous and spontaneous death of all animals during SE observed only at day 9th, suggest that a higher P-gp overexpression in cardiomyocytes could play a role in SUDEP, however, because it is only a study descriptive, further researches are needed to confirm this hypothesis.Fil: Auzmendi, Jerónimo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Merelli, Amalia Margarita. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Girardi, Elena Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Orozco Suárez, Sandra. Centro Médico Nacional Siglo XXI; MéxicoFil: Rocha Arrieta, Luisa Lilia. Centro de Investigación y de Estudios Avanzados. Departamento de Farmacobiología; MéxicoFil: Lazarowski, Alberto Jorge. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Fundación Investigar; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentin

Cannabidiol (Cbd) alters the functionality of neutrophils (pmn). implications in the refractory epilepsy treatment

Cannabidiol (CBD), a lipophilic cannabinoid compound without psychoactive effects, has emerged as adjuvant of anti‐epileptic drugs (AEDs) in the treatment of refractory epilepsy (RE), decreasing the severity and/or frequency of seizures. CBD is considered a multitarget drug that could act throughout the canonical endocannabinoid receptors (CB1‐CB2) or multiple non‐canonical pathways. Despite the fact that the CBD mechanism in RE is still unknown, experiments carried out in our laboratory showed that CBD has an inhibitory role on P‐glycoprotein excretory function, highly related to RE. Since CB2 is expressed mainly in the immune cells, we hypothesized that CBD treatment could alter the activity of polymorphonuclear neutrophils (PMNs) in a similar way that it does with microglia/macrophages and others circulating leukocytes. In vitro, CBD induced PMN cytoplasmatic vacuolization and proapoptotic nuclear condensation, associated with a significantly decreased viability in a concentration‐dependent manner, while low CBD concentration decreased PMN viability in a time‐dependent manner. At a functional level, CBD reduced the chemotaxis and oxygen consumption of PMNs related with superoxide anion production, while the singlet oxygen level was increased suggesting oxidative stress damage. These results are in line with the well-known CBD anti‐inflammatory effect and support a potential immunosuppressor role on PMNs that could promote an eventual defenseless state during chronic treatment with CBD in RE.Fil: Taborda Gómez, Claudia Marcela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Fisiopatología y Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Lairion, Fabiana Norma. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Repetto, Marisa Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Ettcheto, Miren. Universidad de Barcelona; EspañaFil: Merelli, Amalia Margarita. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Fisiopatología y Bioquímica Clínica; ArgentinaFil: Lazarowski, Alberto Jorge. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Fisiopatología y Bioquímica Clínica; ArgentinaFil: Auzmendi, Jerónimo Andrés. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Fisiopatología y Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentin

Ulcerated hemosiderinic dyschromia and iron deposits within lower limbs treated with a topical application of biological chelator

The ulcerative haemosiderinic dyschromia of chronic venous insufficiency is difficult to heal and presents a high accumulation of iron. Lactoferrin, a potent natural iron chelator, could help to scar this ulcerative haemosi - derinic dyschromia. The objective of this study was to determine whether the topical application of a liposomal gel with Lactoferrin favors scarring/degradation of the brown colored spot typical of ulcerative haemosiderinic dyschromia. Nine patients with severe chronic venous insufficiency and ulcerative haemosiderinic dyschromia (CEAP-C6), with a natural evolution of over 12 months, were included in the study. Hemo chromatosis gene mutations were investigated. The levels of serum ferritin, transferrin saturation and blood cell counts were analyzed. The presence of hemosiderin was investigated through periulcerous and ulcer fundus biopsies carried out at baseline and 30 days after treatment with Lactoferrin. The severity of the injuries (CEAP classification) was evaluated at the beginning of and throughout the whole 3-month treatment period. No patient had received compression treatment during the three months previous to this therapy. Significant improvement in these injuries, with a reduction in the dimensions of the brown spot (9 of 9) at Day 90, and complete scarring with a closure time ranging from 15 to 180 days (7 of 9) were observed. The use of topical lactoferrin is a non-invasive therapeutic tool that favors clearance of hemosiderinic dyschromia and scarring of the ulcer. The success of this study was not influenced either by the hemochromatosis genetics or the iron metabolism profile observed