Publications by authors named "Alberto Lazarowski"

50 Publications

From Genome to Drugs: New Approaches in Antimicrobial Discovery.

Front Pharmacol 2021 9;12:647060. Epub 2021 Jun 9.

Instituto de Cálculo, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.

Decades of successful use of antibiotics is currently challenged by the emergence of increasingly resistant bacterial strains. Novel drugs are urgently required but, in a scenario where private investment in the development of new antimicrobials is declining, efforts to combat drug-resistant infections become a worldwide public health problem. Reasons behind unsuccessful new antimicrobial development projects range from inadequate selection of the molecular targets to a lack of innovation. In this context, increasingly available omics data for multiple pathogens has created new drug discovery and development opportunities to fight infectious diseases. Identification of an appropriate molecular target is currently accepted as a critical step of the drug discovery process. Here, we review how diverse layers of multi-omics data in conjunction with structural/functional analysis and systems biology can be used to prioritize the best candidate proteins. Once the target is selected, virtual screening can be used as a robust methodology to explore molecular scaffolds that could act as inhibitors, guiding the development of new drug lead compounds. This review focuses on how the advent of omics and the development and application of bioinformatics strategies conduct a "big-data era" that improves target selection and lead compound identification in a cost-effective and shortened timeline.
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http://dx.doi.org/10.3389/fphar.2021.647060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8219968PMC
June 2021

Cannabidiol (CBD) Alters the Functionality of Neutrophils (PMN). Implications in the Refractory Epilepsy Treatment.

Pharmaceuticals (Basel) 2021 Mar 5;14(3). Epub 2021 Mar 5.

Departamento de Bioquímica Clínica, Facultad de Farmacia y Bioquímica, Instituto de Fisiopatología y Bioquímica Clínica (INFIBIOC), Universidad de Buenos Aires, Buenos Aires C1120AAF, Argentina.

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.
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http://dx.doi.org/10.3390/ph14030220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8001508PMC
March 2021

Myocardial Iron Overload in an Experimental Model of Sudden Unexpected Death in Epilepsy.

Front Neurol 2021 11;12:609236. Epub 2021 Feb 11.

Department of Clinical Biochemistry, School of Pharmacy and Biochemistry, Pathophysiology and Clinical Biochemistry Institute (INFIBIOC), University of Buenos Aires, Buenos Aires, Argentina.

Uncontrolled repetitive generalized tonic-clonic seizures (GTCS) are the main risk factor for sudden unexpected death in epilepsy (SUDEP). GTCS can be observed in models such as Pentylenetetrazole kindling (PTZ-K) or pilocarpine-induced Status Epilepticus (SE-P), which share similar alterations in cardiac function, with a high risk of SUDEP. Terminal cardiac arrhythmia in SUDEP can develop as a result of a high rate of hypoxic stress-induced by convulsions with excessive sympathetic overstimulation that triggers a neurocardiogenic injury, recently defined as "Epileptic Heart" and characterized by heart rhythm disturbances, such as bradycardia and lengthening of the QT interval. Recently, an iron overload-dependent form of non-apoptotic cell death called ferroptosis was described at the brain level in both the PTZ-K and SE-P experimental models. However, seizure-related cardiac ferroptosis has not yet been reported. Iron overload cardiomyopathy (IOC) results from the accumulation of iron in the myocardium, with high production of reactive oxygen species (ROS), lipid peroxidation, and accumulation of hemosiderin as the final biomarker related to cardiomyocyte ferroptosis. Iron overload cardiomyopathy is the leading cause of death in patients with iron overload secondary to chronic blood transfusion therapy; it is also described in hereditary hemochromatosis. GTCS, through repeated hypoxic stress, can increase ROS production in the heart and cause cardiomyocyte ferroptosis. We hypothesized that iron accumulation in the "Epileptic Heart" could be associated with a terminal cardiac arrhythmia described in the IOC and the development of state-potentially in the development of SUDEP. Using the aforementioned PTZ-K and SE-P experimental models, after SUDEP-related repetitive GTCS, we observed an increase in the cardiac expression of hypoxic inducible factor 1α, indicating hypoxic-ischemic damage, and both necrotic cells and hemorrhagic areas were related to the possible hemosiderin production in the PTZ-K model. Furthermore, we demonstrated for the first time an accumulation of hemosiderin in the heart in the SE-P model. These results suggest that uncontrolled recurrent seizures, as described in refractory epilepsy, can give rise to high hypoxic stress in the heart, thus inducing hemosiderin accumulation as in IOC, and can act as an underlying hidden mechanism contributing to the development of a terminal cardiac arrhythmia in SUDEP. Because iron accumulation in tissues can be detected by non-invasive imaging methods, cardiac iron overload in refractory epilepsy patients could be treated with chelation therapy to reduce the risk of SUDEP.
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http://dx.doi.org/10.3389/fneur.2021.609236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7905080PMC
February 2021

Hypoxia, Oxidative Stress, and Inflammation: Three Faces of Neurodegenerative Diseases.

J Alzheimers Dis 2021 ;82(s1):S109-S126

Universidad de Buenos Aires, Facultad de Farmacia y Bioqummica, Departamento de Bioquímica Clínica, Instituto de Fisiopatología y Bioquímica Clínica (INFIBIOC), Argentina.

The cerebral hypoxia-ischemia can induce a wide spectrum of biologic responses that include depolarization, excitotoxicity, oxidative stress, inflammation, and apoptosis, and result in neurodegeneration. Several adaptive and survival endogenous mechanisms can also be activated giving an opportunity for the affected cells to remain alive, waiting for helper signals that avoid apoptosis. These signals appear to help cells, depending on intensity, chronicity, and proximity to the central hypoxic area of the affected tissue. These mechanisms are present not only in a large list of brain pathologies affecting commonly older individuals, but also in other pathologies such as refractory epilepsies, encephalopathies, or brain trauma, where neurodegenerative features such as cognitive and/or motor deficits sequelae can be developed. The hypoxia inducible factor 1α (HIF-1α) is a master transcription factor driving a wide spectrum cellular response. HIF-1α may induce erythropoietin (EPO) receptor overexpression, which provides the therapeutic opportunity to administer pharmacological doses of EPO to rescue and/or repair affected brain tissue. Intranasal administration of EPO combined with other antioxidant and anti-inflammatory compounds could become an effective therapeutic alternative, to avoid and/or slow down neurodegenerative deterioration without producing adverse peripheral effects.
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http://dx.doi.org/10.3233/JAD-201074DOI Listing
January 2021

Supplementation with Resveratrol, Piperine and Alpha-Tocopherol Decreases Chronic Inflammation in a Cluster of Older Adults with Metabolic Syndrome.

Nutrients 2020 Oct 15;12(10). Epub 2020 Oct 15.

Unidad Polifenoles, Vino y Salud, Cuarta Cátedra de Medicina, Hospital de Clínicas "José de San Martín" Facultad de Medicina, Universidad de Buenos Aires, City of Buenos Aires C1120AAF, Argentina.

Metabolic Syndrome (MetS) is increasing worldwide regardless of culture, genetic, gender, and geographic differences. While multiple individual risk factors, such as obesity, hypertension, diabetes, and hyperlipidemia, can cause cardiovascular disease (CVD), it is the intercurrence of these risk factors that defines MetS as a cluster that creates an environment for atherosclerosis and other manifestations of CVD. Despite the advances in the knowledge and management of each of the components of MetS, there are two molecular biology processes, chronic inflammation and oxidative stress, which are still underdiagnosed and undertreated. In order to assess the effect of a dietary supplement on chronic inflammation in MetS, we conducted a clinical trial with volunteers receiving a formula composed of resveratrol, piperine and alpha tocopherol (FRAMINTROL), together with their habitual treatment, for three months. The inflammatory state was evaluated by ultrasensitive C reactive protein (US CRP) and ferritin in plasma, and oxygen consumption and chemiluminescence in neutrophils. The results showed that ferritin decreased by 10% ( < 0.05), US-CRP by 33% ( < 0.0001), oxygen consumption by 55% ( < 0.0001), and spontaneous chemiluminiscence was by 25% ( < 0.005) after treatment. As far as we know, this is the first study showing a chronic inflammation decrease in MetS patients due to the administration of a biopower Resveratrol-piperine and alpha tocopherol dietary supplement together with conventional therapy.
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http://dx.doi.org/10.3390/nu12103149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7602615PMC
October 2020

Cannabidiol (CBD) Inhibited Rhodamine-123 Efflux in Cultured Vascular Endothelial Cells and Astrocytes Under Hypoxic Conditions.

Front Behav Neurosci 2020 17;14:32. Epub 2020 Mar 17.

Instituto de Fisiopatología y Bioquímica Clínica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina.

Despite the constant development of new antiepileptic drugs (AEDs), more than 30% of patients develop refractory epilepsy (RE) characterized by a multidrug-resistant (MDR) phenotype. The "transporters hypothesis" indicates that the mechanism of this MDR phenotype is the overexpression of ABC transporters such as P-glycoprotein (P-gp) in the neurovascular unit cells, limiting access of the AEDs to the brain. Recent clinical trials and basic studies have shown encouraging results for the use of cannabinoids in RE, although its mechanisms of action are still not fully understood. Here, we have employed astrocytes and vascular endothelial cell cultures subjected to hypoxia, to test the effect of cannabidiol (CBD) on the P-gp-dependent Rhodamine-123 (Rho-123) efflux. Results show that during hypoxia, intracellular Rho-123 accumulation after CBD treatment is similar to that induced by the P-gp inhibitor Tariquidar (Tq). Noteworthy, this inhibition is like that registered in non-hypoxia conditions. Additionally, docking studies predicted that CBD could behave as a P-gp substrate by the interaction with several residues in the α-helix of the P-gp transmembrane domain. Overall, these findings suggest a direct effect of CBD on the Rho-123 P-gp-dependent efflux activity, which might explain why the CBD add-on treatment regimen in RE patients results in a significant reduction in seizure frequency.
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http://dx.doi.org/10.3389/fnbeh.2020.00032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090129PMC
March 2020

Paroxysmal nocturnal hemoglobinuria: Test to monitor the action of eculizumab treatment.

Int J Lab Hematol 2020 Jun 23;42(3):335-340. Epub 2020 Mar 23.

Departamento de Bioquímica Clínica, Facultad de Farmacia y Bioquímica, Hospital de Clínicas José de San Martín, Universidad de Buenos Aires (UBA), Buenos Aires, Argentina.

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation in the PIG-A gene, which encodes for glycosylphosphatidylinositol, a phospholipid membrane that anchors proteins like CD55 and CD59. These proteins are inhibitors of the complement-mediated lysis. PNH is diagnosed by flow cytometry, and treatment with eculizumab improves the life quality of patients with severe clinical compromise. The aim of this work was to evaluate a hemolytic test that allows monitoring the blockade of the alternative complement pathway caused by eculizumab (herein MET test).

Methods: There were analyzed a total of 163 serum samples from nine patients with PNH under treatment with eculizumab and ten healthy volunteers like controls. The patients were evaluated for 6 months. The MET test consisted in incubating red blood cells from patients (RBC ) with either acidified serum from healthy volunteers and from patients with PNH. The results can be (a) Positive, (b) Blockade profile, or (c) Negative.

Results: Seven patients responded favorably to the eculizumab, and the test evidenced the blockade profile. The two remaining patients were nonresponders to the treatment, with a positive MET test. In these patients, the dose was increased. One responded favorably with a blockade profile, and the other continued to be nonresponder.

Conclusions: The MET test proved to be a useful tool to monitor the blockade of complement by eculizumab.
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http://dx.doi.org/10.1111/ijlh.13186DOI Listing
June 2020

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

Epilepsia Open 2020 Mar 17;5(1):36-49. Epub 2020 Jan 17.

Departamento de Bioquímica Clínica Facultad de Farmacia y Bioquímica Instituto de Investigaciones en Fisiopatología y Bioquímica Clínica (INFIBIOC) Universidad de Buenos Aires Buenos Aires Argentina.

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.
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http://dx.doi.org/10.1002/epi4.12376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049809PMC
March 2020

EPO and EPO-Receptor System as Potential Actionable Mechanism for the Protection of Brain and Heart in Refractory Epilepsy and SUDEP.

Curr Pharm Des 2020 ;26(12):1356-1364

Universidad de Buenos Aire (UBA), Facultad de Farmacia y Bioquimica (FFyB), Instituto de Fisiopatologia y Bioquimica Clínica (INFIBIOC), Junín 956, Ciudad Autonoma de Buenos Aires (CABA), Buenos Aires, Argentina.

The most important activity of erythropoietin (EPO) is the regulation of erythrocyte production by activation of the erythropoietin receptor (EPO-R), which triggers the activation of anti-apoptotic and proliferative responses of erythroid progenitor cells. Additionally, to erythropoietic EPO activity, an antiapoptotic effect has been described in a wide spectrum of tissues. EPO low levels are found in the central nervous system (CNS), while EPO-R is expressed in most CNS cell types. In spite of EPO-R high levels expressed during the hypoxicischemic brain, insufficient production of endogenous cerebral EPO could be the cause of determined circuit alterations that lead to the loss of specific neuronal populations. In the heart, high EPO-R expression in cardiac progenitor cells appears to contribute to myocardial regeneration under EPO stimulation. Several lines of evidence have linked EPO to an antiapoptotic role in CNS and in heart tissue. In this review, an antiapoptotic role of EPO/EPO-R system in both brain and heart under hypoxic conditions, such as epilepsy and sudden death (SUDEP) has been resumed. Additionally, their protective effects could be a new field of research and a novel therapeutic strategy for the early treatment of these conditions and avoid SUDEP.
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http://dx.doi.org/10.2174/1381612826666200219095548DOI Listing
November 2020

Acquisition of stem associated-features on metastatic osteosarcoma cells and their functional effects on mesenchymal stem cells.

Biochim Biophys Acta Gen Subj 2020 04 13;1864(4):129522. Epub 2020 Jan 13.

Remodeling processes and cellular niches laboratory, Instituto de Medicina Traslacional e Ingeniería Biomédica (IMTIB), CONICET- Hospital Italiano Buenos Aires (HIBA), Instituto Universitario del Hospital Italiano (IUHI), C1199ACL Buenos Aires, Argentina. Electronic address:

Background: Osteosarcoma (OS) is the most frequent malignant bone tumor, affecting predominantly children and young adults. Metastases are a major clinical challenge in OS. In this context, 20% of OS patients are diagnosed with metastatic OS, but near 80% of all OS patients could present non-detectable micrometastases at the moment of diagnosis.

Methods: Osteogenic differentiation; doxorubicin exclusion assay; fluorescence microscopy; RT-qPCR; proteomic analysis.

Results: Our results suggest that metastatic OS cells possess a diminished osteoblastic differentiation potential with a gain of metastatic traits like the capacity to modify intracellular localization of chemodrugs and higher levels of expression of stemness-related genes. On the opposite hand, non-metastatic OS cells possess bone-associated traits like higher osteoblastic differentiation and also an osteoblastic-inducer secretome. OS cells also differ in the nature of their interaction with mesenchymal stem cells (MSCs), with opposites impacts on MSCs phenotype and behavior.

Conclusions: All this suggests that a major trait acquired by metastatic cells is a switch into a stem-like state that could favor its survival in the pulmonary niche, opening new possibilities for personalized chemotherapeutic schemes.

General Significance: Our work provides new insights regarding differences among metastatic and non-metastatic OS cells, with particular emphasis on differentiation potential, multidrug resistance and interaction with MSCs.
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http://dx.doi.org/10.1016/j.bbagen.2020.129522DOI Listing
April 2020

The role of P-glycoprotein (P-gp) and inwardly rectifying potassium (Kir) channels in sudden unexpected death in epilepsy (SUDEP).

Epilepsy Behav 2021 08 6;121(Pt B):106590. Epub 2019 Nov 6.

INFIBIOC, Departamento de Bioquímica Clínica, Facultad de Farmacia y Bioquímica (FFyB), Universidad de Buenos Aires (UBA), Buenos Aires, Argentina. Electronic address:

Sudden unexpected death in epilepsy (SUDEP) is the major cause of death that affects patients with epilepsy. The risk of SUDEP increases according to the frequency and severity of uncontrolled seizures; therefore, SUDEP risk is higher in patients with refractory epilepsy (RE), in whom most antiepileptic drugs (AEDs) are ineffective for both seizure control and SUDEP prevention. Consequently, RE and SUDEP share a multidrug resistance (MDR) phenotype, which is mainly associated with brain overexpression of ABC-transporters such as P-glycoprotein (P-gp). The activity of P-gp can also contribute to membrane depolarization and affect the normal function of neurons and cardiomyocytes. Other molecular regulators of membrane potential are the inwardly rectifying potassium channels (Kir), whose genetic variants have been related to both epilepsy and heart dysfunctions. Although it has been suggested that dysfunctions of the cardiac, respiratory, and brainstem arousal systems are the causes of SUDEP, the molecular basis for explaining its dysfunctions remain unknown. In rats, repetitive seizures or status epilepticus induced high expression of P-gp and loss Kir expression in the brain and heart, and promoted membrane depolarization, malignant bradycardia, and the high rate of mortality. Here we reviewed clinical and experimental evidences suggesting that abnormal expression of depolarizing/repolarizing factors as P-gp and Kir could favor persistent depolarization of membranes without any rapid functional recovery capacity. This condition induced by convulsive stress could be the molecular mechanism leading to acquired severe bradycardia, as an ineffective heart response generating the appropriate scenario for SUDEP development. This article is part of the Special Issue "NEWroscience 2018".
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http://dx.doi.org/10.1016/j.yebeh.2019.106590DOI Listing
August 2021

New anticonvulsant candidates prevent P-glycoprotein (P-gp) overexpression in a pharmacoresistant seizure model in mice.

Epilepsy Behav 2021 08 13;121(Pt B):106451. Epub 2019 Aug 13.

Laboratorio de Investigación y Desarrollo de Bioactivos (LIDeB), Departamento de Ciencias Biologicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata (UNLP), 47 y115, La Plata B1900BJW, Argentina. Electronic address:

Despite the approval of a considerable number of last generation antiepileptic drugs (AEDs) (only in the last decade, six drugs have gained Food and Drug Administration approval), the global figures of seizure control have seemingly not improved, and available AED can still be regarded as symptomatic treatments. Fresh thinking in AEDs drug discovery, including the development of drugs with novel mechanisms of action, is required to achieve truly innovative antiepileptic medications. The transporter hypothesis proposes that inadequate penetration of AEDs across the blood-brain barrier, caused by increased expression of efflux transporters such as P-glycoprotein (P-gp), contributes to drug-resistant epilepsy. Neuroinflammation due to high levels of glutamate has been identified as one of the causes of P-gp upregulation, and several studies in animal models of epilepsy suggest that antiinflammatory drugs might prevent P-gp overexpression and, thus, avoid the development of refractory epilepsy. We have applied ligand-based in silico screening to select compounds that exert dual anticonvulsant and antiinflammatory effects. Five of the hits were tested in animal models of seizure, with protective effects. Later, two of them (sebacic acid (SA) and gamma-decanolactone) were submitted to the recently described MP23 model of drug-resistant seizures. All in all, SA displayed the best profile, showing activity in the maximal electroshock seizure (MES) and pentylenetetrazol (PTZ) seizure models, and reversing resistance to phenytoin (PHT) and decreasing the P-gp upregulation in the MP23 model. Furthermore, pretreatment with SA in the pilocarpine status epilepticus (SE) model resulted in decreased histamine release in comparison with nontreated animals. This is the first report of the use of the MP23 model to screen for novel anticonvulsant compounds that may avoid the development of P-gp-related drug resistance.
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http://dx.doi.org/10.1016/j.yebeh.2019.106451DOI Listing
August 2021

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

Front Neurosci 2019 17;13:750. Epub 2019 Jul 17.

Laboratorio de Neuropatología Molecular, Instituto de Biología Celular y Neurociencia "Prof. E. De Robertis" IBCN-UBA-CONICET, Universidad de Buenos Aires, Buenos Aires, Argentina.

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. , 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 CoCl (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 3 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.
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http://dx.doi.org/10.3389/fnins.2019.00750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6652211PMC
July 2019

Metastatic Niches and the Modulatory Contribution of Mesenchymal Stem Cells and Its Exosomes.

Int J Mol Sci 2019 Apr 20;20(8). Epub 2019 Apr 20.

Remodelative Processes and Cellular Niches Laboratory, Instituto de Medicina Traslacional e Ingeniería Biomédica (IMTIB)-CONICET-Instituto Universitario del Hospital Italiano-Hospital Italiano Buenos Aires (HIBA), C1199ACL Buenos Aires, Argentina.

Mesenchymal stem cells (MSCs) represent an interesting population due to their capacity to release a variety of cytokines, chemokines, and growth factors, and due to their motile nature and homing ability. MSCs can be isolated from different sources, like adipose tissue or bone marrow, and have the capacity to differentiate, both in vivo and in vitro, into adipocytes, chondrocytes, and osteoblasts, making them even more interesting in the regenerative medicine field. Tumor associated stroma has been recognized as a key element in tumor progression, necessary for the biological success of the tumor, and MSCs represent a functionally fundamental part of this associated stroma. Exosomes represent one of the dominant signaling pathways within the tumor microenvironment. Their biology raises high interest, with implications in different biological processes involved in cancer progression, such as the formation of the pre-metastatic niche. This is critical during the metastatic cascade, given that it is the formation of a permissive context that would allow metastatic tumor cells survival within the new environment. In this context, we explored the role of exosomes, particularly MSCs-derived exosomes as direct or indirect modulators. All this points out a possible new tool useful for designing better treatment and detection strategies for metastatic progression, including the management of chemoresistance.
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http://dx.doi.org/10.3390/ijms20081946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6515194PMC
April 2019

High incidence of persistent subtherapeutic levels of the most common AEDs in children with epilepsy receiving polytherapy.

Epilepsy Res 2018 12 22;148:107-114. Epub 2018 Sep 22.

INFIBIOC-FFyB-UBA, Junín 954, 1113, CABA, Argentina; Fundación Investigar, C. Beliera 3025 (RN8), B1629WWA Pilar, Buenos Aires, Argentina. Electronic address:

Background: Low levels of AEDs can be secondary drug-drug interactions or related to irregular intake due to poor treatment adherence. This latter behavior is highly suspected in ambulatory pediatric epileptic patients when controls of AEDs are subtherapeutic. However, it cannot be considered for inpatients during long periods of hospitalization. A few isolated case reports have documented persistent low levels (PLL) of AEDs in hospitalized epileptic children, but no population study has currently been reported.

Objective: The aim of this study was to document the incidence of PLL of the most common AEDs - phenytoin (PHT), phenobarbital (PHB), valproic acid (VA), and carbamazepine (CBZ) - in pediatric epileptic in- and outpatients (PEP).

Methods: 21,040 plasma levels of the aforementioned AEDs from 3279 PEP were retrospectively analyzed. Plasma levels of AEDs were measured by an automated method using trademarked commercial kits with their corresponding quality control programs. Randomized samples were also controlled by HPLC methods. Only cases with more than 3 controls were included in the study.

Results: A high rate of PLL of PHT was detected in in- (71.7%) and outpatients (74.1%), while PLL of PHB, VA, and CBZ were detected in a lower proportion. Rates of PLL of PHT were similar in in- and outpatients. PLL of PHB was more commonly observed in outpatients while PLL of VA and CBZ were more frequently seen in inpatients. In some hospitalized patients receiving polytherapy, PLL of at least one AED were documented during a long time.

Discussion: Treatment non-adherence could be present in part of the outpatients, but cannot explain the PLL observed in a group of inpatients as described here. The recently described "pharmacokinetic hypothesis" of pharmaco-resistant epilepsy should be addressed in cases with AEDs-PLL, particularly in hospitalized cases. Perhaps, instead of stopping the subtherapeutic medication, the increasing doses of this AED and/or administration of inhibitors of CYP and P-glycoprotein, could help to achieve its therapeutic range, allowing a better pharmacologic effect and avoiding the development of more severe complications, such as status epilepticus or SUDEP.
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http://dx.doi.org/10.1016/j.eplepsyres.2018.09.008DOI Listing
December 2018

Pilocarpine-Induced Status Epilepticus Is Associated with P-Glycoprotein Induction in Cardiomyocytes, Electrocardiographic Changes, and Sudden Death.

Pharmaceuticals (Basel) 2018 Feb 16;11(1). Epub 2018 Feb 16.

Departamento de Bioquímica Clínica, Instituto de Investigaciones en Fisiopatología y Bioquímica Clínica (INFIBIOC), Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Junín 956, Buenos Aires C1113AAD, Argentina.

Sudden unexpected death in epilepsy (SUDEP) is the major cause of death in those patients suffering from refractory epilepsy (RE), with a 24-fold higher risk relative to the normal population. SUDEP risk increases with seizure frequency and/or seizure-duration as in RE and Status Epilepticus (SE). P-glycoprotein (P-gp), the product of the multidrug resistant gene, is a detoxifying pump that extrudes drugs out of the cells and can confer pharmacoresistance to the expressing cells. Neurons and cardiomyocytes normally do not express P-gp, however, it is overexpressed in the brain of patients or in experimental models of RE and SE. P-gp was also detected after brain or cardiac hypoxia. We have previously demonstrated that repetitive pentylenetetrazole (PTZ)-induced seizures increase P-gp expression in the brain, which is associated with membrane depolarization in the hippocampus, and in the heart, which is associated with fatal SE. SE can produce hypoxic-ischemic altered cardiac rhythm (HIACR) and severe arrhythmias, and both are related with SUDEP. Here, we investigate whether SE induces the expression of hypoxia-inducible transcription factor (HIF)-1α and P-gp in cardiomyocytes, which is associated with altered heart rhythm, and if these changes are related with the spontaneous death rate. SE was induced in Wistar rats once a week for 3 weeks, by lithium-pilocarpine-paradigm. Electrocardiograms, HIF-1α, and P-gp expression in cardiomyocytes, were evaluated in basal conditions and 72 h after SE. All spontaneous deaths occurred 48 h after each SE was registered. We observed that repeated SE induced HIF-1α and P-gp expression in cardiomyocytes, electrocardiographic (ECG) changes, and a high rate of spontaneous death. Our results suggest that the highly accumulated burden of convulsive stress results in a hypoxic heart insult, where P-gp expression may play a depolarizing role in cardiomyocyte membranes and in the development of the ECG changes, such as QT interval prolongation, that could be related with SUDEP. We postulate that this mechanism could explain, in part, the higher SUDEP risk in patients with RE or SE.
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http://dx.doi.org/10.3390/ph11010021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874717PMC
February 2018

Understanding the Role of Hypoxia Inducible Factor During Neurodegeneration for New Therapeutics Opportunities.

Curr Neuropharmacol 2018 ;16(10):1484-1498

INFIBIOC-FFyB-UBA-Buenos, Aires, Argentina.

Neurodegeneration (NDG) is linked with the progressive loss of neural function with intellectual and/or motor impairment. Several diseases affecting older individuals, including Alzheimer's disease, Amyotrophic Lateral Sclerosis, Huntington's disease, Parkinson's disease, stroke, Multiple Sclerosis and many others, are the most relevant disorders associated with NDG. Since other pathologies such as refractory epilepsy, brain infections, or hereditary diseases such as "neurodegeneration with brain iron accumulation", also lead to chronic brain inflammation with loss of neural cells, NDG can be said to affect all ages. Owing to an energy and/or oxygen supply imbalance, different signaling mechanisms including MAPK/PI3K-Akt signaling pathways, glutamatergic synapse formation, and/or translocation of phosphatidylserine, might activate some central executing mechanism common to all these pathologies and also related to oxidative stress. Hypoxia inducible factor 1-α (HIF-1α) plays a twofold role through gene activation, in the sense that this factor has to "choose" whether to protect or to kill the affected cells. Most of the afore-mentioned processes follow a protracted course and are accompanied by progressive iron accumulation in the brain. We hypothesize that the neuroprotective effects of iron chelators are acting against the generation of free radicals derived from iron, and also induce sufficient -but not excessive- activation of HIF-1α, so that only the hypoxia-rescue genes will be activated. In this regard, the expression of the erythropoietin receptor in hypoxic/inflammatory neurons could be the cellular "sign" to act upon by the nasal administration of pharmacological doses of Neuro-EPO, inducing not only neuroprotection, but eventually, neurorepair as well.
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http://dx.doi.org/10.2174/1570159X16666180110130253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6295932PMC
November 2018

Role of JNK isoforms in the kainic acid experimental model of epilepsy and neurodegeneration.

Front Biosci (Landmark Ed) 2017 01 1;22:795-814. Epub 2017 Jan 1.

Unitat de Farmacologia i Farmacognosia, Facultat de Farmacia, Universitat de Barcelona, Barcelona, Spain, and Biomedical Research Networking Center in Neurodegenerative Diseases (CIBERNED), Madrid, Spain,

Chemoconvulsants that induce status epilepticus in rodents have been widely used over the past decades due to their capacity to reproduce with high similarity neuropathological and electroencephalographic features observed in patients with temporal lobe epilepsy (TLE). Kainic acid  is one of the most used chemoconvulsants in experimental models. KA administration mainly induces neuronal loss in the hippocampus. We focused the present review inthe c-Jun N-terminal kinase-signaling pathway (JNK), since it has been shown to play a key role in the process of neuronal death following KA activation. Among the three isoforms of JNK (JNK1, JNK2, JNK3), JNK3 is widely localized in the majority of areas of the hippocampus, whereas JNK1 levels are located exclusively in the CA3 and CA4 areas and in dentate gyrus. Disruption of the gene encoding JNK3 in mice renders neuroprotection to KA, since these animals showed a reduction in seizure activity and a diminution in hippocampal neuronal apoptosis. In light of this, JNK3 could be a promising subcellular target for future therapeutic interventions in epilepsy.
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http://dx.doi.org/10.2741/4517DOI Listing
January 2017

MDR1/ABCB1 gene polymorphisms in patients with chronic myeloid leukemia.

Blood Res 2015 Sep 22;50(3):154-9. Epub 2015 Sep 22.

INFIBIOC-FFyB-UBA, Buenos Aires, Argentina. ; Fundación Investigar, Buenos Aires, Argentina.

Background: Tyrosine kinase inhibitors (TKIs) are the recommended treatment for patients with chronic myeloid leukemia (CML). The MDR1/ABCB1 gene plays a role in resistance to a wide spectrum of drugs, including TKIs. However, the association of MDR1/ABCB1 gene polymorphisms (SNPs) such as C1236T, G2677T/A, and C3435T with the clinical therapeutic evolution of CML has been poorly studied. We investigated these gene polymorphisms in CML-patients treated with imatinib, nilotinib and/or dasatinib.

Methods: ABCB1-SNPs were studied in 22 CML-patients in the chronic phase (CP) and 2 CML-patients in blast crisis (BC), all of whom were treated with TKIs, and compared with 25 healthy controls using nested-PCR and sequencing techniques.

Results: Seventeen different haplotypes were identified: 7 only in controls, 6 only in CML-patients, and the remaining 4 in both groups. The distribution ratios of homozygous TT-variants present on each exon between controls and CML-patients were 2.9 for exon 12, and 0.32 for the other 2 exons. Heterozygous T-variants were observed in all controls (100%) and 75% of CML-patients. Wt-haplotype (CC-GG-CC) was observed in 6 CML-patients (25%). In this wt-group, two were treated with nilotinib and reached a major molecular response. The remaining 4 cases had either a minimal or null molecular response, or developed bone marrow aplasia.

Conclusion: Our results suggest that SNPs of the MDR1/ABCB1 gene could help to characterize the prognosis and the clinical-therapeutic evolution of CML-patients treated with TKIs. Wt-haplotype could be associated with a higher risk of developing CML, and a worse clinical-therapeutic evolution.
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http://dx.doi.org/10.5045/br.2015.50.3.154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4595581PMC
September 2015

Combinational Effect of CYP3A5 and MDR-1 Polymorphisms on Tacrolimus Pharmacokinetics in Liver Transplant Patients.

Exp Clin Transplant 2015 Oct;13(5):441-8

Grupo de Investigación en Farmacología y Toxicología (INFARTO), Centro de Información y Estudio de Medicamentos y Tóxicos (CIEMTO), University of Antioquia, Colombia.

Objectives: Previous studies have reported reduced tacrolimus dose-adjusted exposure in individuals expressing the CYP3A5*1 allele (reference single nucleotide polymorphism identification number 776746). However, results on patients from South America are scarce. The objective of this study was to investigate the influence of CYP3A5 and MDR1 allelic variants and their correlation on the pharmacokinetics of tacrolimus and a modified release formulation of tacrolimus in stable patients with liver transplant.

Materials And Methods: This was a prospective, single center, open-label study. Included patients were ≥ 18 years old and receiving a stable dose of tacrolimus for at least 6 months. Patients receiving stable treatment of twice daily tacrolimus were switched to a once-daily dose of modified release tacrolimus, on a milligram-to-milligram basis, with the modified release formulation administered for at least 4 weeks. Blood levels of tacrolimus were obtained before and 1 month after treatment was switched to the modified release formulation.

Results: The frequency of the intron 3 allelic variant of the CYP3A5 isoform (G-to-A substitution at nucleotide 6986) in recipients was 16.6% and 25% in donors. Dose levels of tacrolimus and the modified formulation were significantly higher in donors and recipients who expressed CYP3A5 versus donors and recipients who did not express this allele. In addition, patients who received a liver from a donor expressing CYP3A5 had significantly lower trough concentrations of tacrolimus and the modified formulation. CYP3A5 expression in the donor liver affected tacrolimus (40.46%, P = .001) and modified formulation (37.56%, P = .001) variability. No association was found between the ABCB1 genotype and levels of tacrolimus or its modified formulation.

Conclusions: Our data suggest that CYP3A5*1 in either the donor or recipient resulted in higher mean daily doses of tacrolimus or its modified formulation to achieve target drug exposure in liver transplant patients.
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October 2015

Twenty-One Novel EGFR Kinase Domain variants in Patients with Nonsmall Cell Lung Cancer.

Ann Hum Genet 2015 Nov 29;79(6):385-93. Epub 2015 Sep 29.

Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Roque Saenz Peña 352, Bernal, (B1876BXD), Buenos Aires, Argentina.

Somatic sequence variants in the epidermal growth factor receptor (EGFR) kinase domain are associated with sensitivity to tyrosine kinase inhibitors (TKIs) in patients with nonsmall cell lung cancer (NSCLC). Patients exhibiting sequence variants in this domain that produce kinase activity enhancement, are more likely to benefit from TKIs than patients with EGFR wild-type disease. Although most NSCLC EGFR-related alleles are concentrated in a few positions, established protocols recommend sequencing EGFR exons 18-21. In this study, 21 novel somatic variants belonging to such exons in adult Argentinean patients affected with NSCLC are reported. Of these, 18 were single amino acid substitutions (SASs), occurring alone or in combination with another genetic alteration (complex cases), one was a short deletion, one was a short deletion-short insertion combination, and one was a duplication. New variants and different combinations of previously reported variants were also found. Moreover, two of the reported SASs occurred in previously unreported positions of the EGFR kinase domain. In order to characterize the new sequence variants, physicochemical, sequence and conformational analyses were also performed. A better understanding of sequence variants in NSCLC may facilitate the most appropriate treatment choice for this complex disease.
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http://dx.doi.org/10.1111/ahg.12127DOI Listing
November 2015

Masitinib for the treatment of mild to moderate Alzheimer's disease.

Expert Rev Neurother 2015 Jun 11;15(6):587-96. Epub 2015 May 11.

Unitat de Bioquimica i Biotecnología, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, Reus, Tarragona, Spain.

Alzheimer's disease (AD) is a degenerative neurological disorder that is the most common cause of dementia and disability in older patients. Available treatments are symptomatic in nature and are only sufficient to improve the quality of life of AD patients temporarily. A potential strategy, currently under investigation, is to target cell-signaling pathways associated with neurodegeneration, in order to decrease neuroinflammation, excitotoxicity, and to improve cognitive functions. Current review centers on the role of neuroinflammation and the specific contribution of mast cells to AD pathophysiology. The authors look at masitinib therapy and the evidence presented through preclinical and clinical trials. Dual actions of masitinib as an inhibitor of mast cell-glia axis and a Fyn kinase blocker are discussed in the context of AD pathology. Masitinib is in Phase III clinical trials for the treatment of malignant melanoma, mastocytosis, multiple myeloma, gastrointestinal cancer and pancreatic cancer. It is also in Phase II/III clinical trials for the treatment of multiple sclerosis, rheumatoid arthritis and AD. Additional research is warranted to better investigate the potential effects of masitinib in combination with other drugs employed in AD treatment.
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http://dx.doi.org/10.1586/14737175.2015.1045419DOI Listing
June 2015

Mavoglurant as a treatment for Parkinson's disease.

Expert Opin Investig Drugs 2014 Aug 24;23(8):1165-79. Epub 2014 Jun 24.

Universitat de Barcelona, Institut de Biomedicina (IBUB), Centros de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED), Unitat de Farmacologia I Farmacognòsia, Facultat de Farmàcia , Barcelona, Avda/Joan XXIII , Spain

Introduction: A major unresolved issue in the Parkinson's disease (PD) treatment is the development of l-DOPA-induced dyskinesias (LIDs) as a side effect of chronic L-DOPA administration. Currently, LIDs are managed in part by reducing the L-DOPA dose or by the administration of amantadine. However, this treatment is only partially effective. A potential strategy, currently under investigation, is the coadministration of metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulators (NAMs) and L-DOPA; a treatment that results in the improvement of dyskinesia symptoms and that permits reductions in l-DOPA dosage frequency.

Areas Covered: The authors examine the role of mGluR5 in the pathophysiology of PD and the potential use of mGluR5 NAM as an adjuvant therapy together with a primary treatment with L-DOPA. Specifically, the authors look at the mavoglurant therapy and the evidence presented through preclinical and clinical trials.

Expert Opinion: Interaction between mGluR5 NAM and L-DOPA is an area of interest in PD research as concomitant treatment results in the improvement of LID symptoms in humans, thus enhancing the patient's quality of life. However, few months ago, Novartis decided to discontinue clinical trials of mavoglurant for the treatment of LID, due to the lack of efficacy demonstrated in trials NCT01385592 and NCT01491529, although no safety concerns were involved in this decision. Nevertheless, the potential application of mGluR5 antagonists as neuroprotective agents must be considered and further studies are warranted to better investigate their potential.
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http://dx.doi.org/10.1517/13543784.2014.931370DOI Listing
August 2014

ABC-transporters as stem-cell markers in brain dysplasia/tumor epilepsies.

Front Biosci (Landmark Ed) 2014 Jun 1;19:1425-35. Epub 2014 Jun 1.

Servicio de Neurologia, Hospital Juan P. Garraham, Combate de Los Pozos 1881(1245), CABA, Argentina.

ABC-transporters prevent the access of antiepileptic drugs into brain parenchyma, which partly explains why seizures are frequently refractory to AEDs treatment. Overexpression of ABC-transporters and stem-cell markers including CD34, have been detected in malformations of cortical development (MCD) and brain tumors. ABC-transporters are constitutively expressed during maturation of normal progenitor stem-cells and cancer stem-cells. These abnormal/immature cells of MCD or brain tumors play an active role in the epileptogenesis but the precise nature of this phenomenon is unclear. Irrespective of their property in the pharmacoresistance, ABCB1-transporter P-glycoprotein also plays a role in the membrane depolarization, suggesting that constitutive P-glycprotein overexpression in MCD and brain tumors could explain their epileptogeneic properties. MCD as wells as brain tumors arise from abnormal progenitor cells, where ABC-t together with others stem cell markers, could help to better identification of these abnormal progenitor cells and serve as biomarker of risk for seizure relapse after epilepsy surgery.
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http://dx.doi.org/10.2741/4293DOI Listing
June 2014

Pharmacoresistant epilepsy and nanotechnology.

Front Biosci (Elite Ed) 2014 Jun 1;6:329-40. Epub 2014 Jun 1.

Department of Pharmacobiology, Center of Research , Advanced Studies, Mexico.

Epilepsy is one of the most common chronic neurological disorders. Furthermore, it is associated to diminished health-related quality of life and is thus considered a major public health problem. In spite of the large number of available and ongoing development of several new antiepileptic drugs (AEDs), a high percentage of patients with epilepsy (35-40%) are resistant to pharmacotherapy. A hypothesis to explain pharmacoresistance in epilepsy suggests that overexpression of multidrug resistance proteins, such as P-glycoprotein, on the endothelium of the blood brain barrier represents a challenge for effective AED delivery and concentration levels in the brain. Proven therapeutic strategies to control pharmacoresistant epilepsy include epilepsy surgery and neuromodulation. Unfortunately, not all patients are candidates for these therapies. Nanotechnology represents an attractive strategy to overcome the limited brain access of AEDs in patients with pharmacoresistant epilepsy. This manuscript presents a review of evidences supporting this idea.
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http://dx.doi.org/10.2741/709DOI Listing
June 2014

Genetic variants in C5 and poor response to eculizumab.

N Engl J Med 2014 Feb;370(7):632-9

From Osaka University Graduate School of Medicine (J.N., M.Y., H.S., Y.K.) and World Premier International Immunology Frontier Research Center and Research Institute for Microbial Diseases, Osaka University, Suita (T.K.), Medical Research Institute and Hard Tissue Genome Research Center, Tokyo Medical and Dental University (S.H., J.I.), Tokyo Medical University (K.O.), and Alexion Pharma (M.H.), Tokyo, Tokai University School of Medicine, Isehara (K.A.), Fukushima Medical University, Fukushima (H.N., T.S.), Ichinomiya Municipal Hospital, Ichinomiya (K.K.), Hamanomachi Hospital, Fukuoka (T.E.), Yamaguchi Grand Medical Center, Hofu (T.T.), Niigata University Medical and Dental Hospital, Niigata (M.M.), Japanese Red Cross Takayama Hospital, Takayama (T.M.), and Iwate Prefectual Central Hospital, Morioka (Y.W.) - all in Japan; University of Buenos Aires, Buenos Aires (A.L.B., A.L.); and Alexion Pharmaceuticals, Cheshire, CT (L.L., K.J., P.T.).

Background: Eculizumab is a humanized monoclonal antibody that targets complement protein C5 and inhibits terminal complement-mediated hemolysis associated with paroxysmal nocturnal hemoglobinuria (PNH). The molecular basis for the poor response to eculizumab in a small population of Japanese patients is unclear.

Methods: We assessed the sequences of the gene encoding C5 in patients with PNH who had either a good or poor response to eculizumab. We also evaluated the functional properties of C5 as it was encoded in these patients.

Results: Of 345 Japanese patients with PNH who received eculizumab, 11 patients had a poor response. All 11 had a single missense C5 heterozygous mutation, c.2654G → A, which predicts the polymorphism p.Arg885His. The prevalence of this mutation among the patients with PNH (3.2%) was similar to that among healthy Japanese persons (3.5%). This polymorphism was also identified in a Han Chinese population. A patient in Argentina of Asian ancestry who had a poor response had a very similar mutation, c.2653C → T, which predicts p.Arg885Cys. Nonmutant and mutant C5 both caused hemolysis in vitro, but only nonmutant C5 bound to and was blocked by eculizumab. In vitro hemolysis due to nonmutant and mutant C5 was completely blocked with the use of N19-8, a monoclonal antibody that binds to a different site on C5 than does eculizumab.

Conclusions: The functional capacity of C5 variants with mutations at Arg885, together with their failure to undergo blockade by eculizumab, account for the poor response to this agent in patients who carry these mutations. (Funded by Alexion Pharmaceuticals and the Ministry of Health, Labor, and Welfare of Japan.).
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http://dx.doi.org/10.1056/NEJMoa1311084DOI Listing
February 2014

Evaluation of hypoxia inducible factor expression in inflammatory and neurodegenerative brain models.

Int J Biochem Cell Biol 2013 Jul 17;45(7):1377-88. Epub 2013 Apr 17.

Unitat de Farmacologia I Farmacognòsia, Facultat de Farmàcia, Institut de Biomedicina, IBUB, Centros de Investigación Biomédica en Red de Enfermedades Neurodegenerativas, Universitat de Barcelona, Spain.

The neuroinflammatory process is thought to contribute to the progression of neurological disorders and brain pathologies. The release of pro-inflammatory cytokines and chemokines by activated glial cells, astrocytes and microglia plays an important role in this process. However, the role of hypoxia-inducible factor-1α (HIF-1α), the key transcription factor regulating the expression of hypoxia-inducible genes, during glial activation is less known. Thus, we examined the significance of HIF-1α in three experimental models: first in an acute model of inflammation induced by pro-inflammatory cytokines TNF-α, IL-1β and IFN-γ; secondly, in a chronic model of inflammation using an APPswe/PS1dE9 (APP/PS1) transgenic mouse model of Alzheimer's disease and thirdly via the inhibition of the PI3K/AKT pathway in a model of neuronal apoptosis. During acute glial inflammation induced by in vitro administration of TNF-α, IL-1β and IFN-γ, mRNA expression levels of HIF-1α were significantly upregulated; however, this effect was blocked by SP600126, a pharmacological inhibitor of mitogen-activated protein kinases (MAPKs). These data suggest that MAPKs could be involved in HIF-1α regulation. In addition, we observed that HIF-1α is not involved in the neuronal apoptotic process mediated by PI3-kinase inhibition, which is regulated by c-Jun. Finally, we did not detect significant differences in the expression of HIF-1α mRNA in APP/PS1 mice during the course of the study (3-12 months of age). Thus, we demonstrated that HIF-1α has a prominent role in acute but not in chronic inflammatory processes, such as the one which occurs in the APP/PS1 experimental model of AD. Moreover, HIF-1α is not involved in neuronal apoptosis after PI3K/AKT inhibition.
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http://dx.doi.org/10.1016/j.biocel.2013.04.011DOI Listing
July 2013

Usefulness of salivary drug monitoring for detecting efflux transporter overexpression.

Curr Pharm Des 2013 ;19(38):6701-8

Pharmaceutical Sciences Department, Faculty of Chemistry, P.O.Box 1157., 11800 Montevideo, Uruguay.

Background: Bioavailability (F) and clearance (CL) are two pharmacokinetic parameters difficult to differentiate from simple plasma measurement when a drug is administered orally. Venous (V) / artery (A) concentration ratio of a drug could be a reliable index of its CL if measurements of plasma concentration were performed during a period of time where the absorption process was not longer operative, then during a pure elimination phase.

Objective: A novel subrogate using two protocolized saliva samples sequentially collected (first, S1, and second, S2) was designed in order to replace V and A free plasma drug concentrations, respectively. Two drugs, phenytoin (PHT) and carbamazepine (CBZ), which are well-known for their inducer properties and their dose-dependent clearance variations, were studied taking into account the sex of individuals.

Setting And Patients: A multicentre two-phase collaborative study was done. The first phase was performed with healthy volunteers in order to determine salivary pharmacokinetic parameters after single dose administration. Twelve volunteers (6 male and 6 female) received 400 mg of CBZ (2 tablets x 200 mg, immediate release product). Twenty four volunteers (10 male and 14 female) received 100 mg of PHT. The second phase was carried out with epileptic patients under CBZ (11 male 15 female) or PHT (11 male and 11 female) monotherapy, in order to study dose-related and sex-related pharmacokinetic differences.

Main Outcome Measures: In the single dose trials, peaks (Tmax, Cmax) were computed directly from the data. Areas under concentrationtime curves (AUC∞), AUC∞xW (area corrected by weight) and half-lives (t1/2) were calculated. In the case of CBZ, AUCCBZ-10,11- epoxide/AUCCBZ metabolic ratios were also calculated. After multiple dose administration, S1 and S2 trough morning drug concentrations were measured.

Results: Cmax and AUC differed significantly between sexes for the two drugs after single dose administration. Nevertheless, the apparent clearance (CL/F) per unit of body weight did not differ (CBZ) or slightly differed (PHT) between sexes. Higher metabolic ratio for CBZ in women would lead to lower F and therefore lower CL in this gender. In the case of PHT, women would have either lower F or higher CL than men. After multiple dose administration, S1/S2 saliva drug concentration ratio correlated positively with S2 for CBZ, showing that CBZ clearance increases with daily dose. Gender differences were also observed for CBZ-10,11-epoxide concentration, being bioavailability the main parameter responsible for this difference. S1/S2 saliva PHT concentration ratio correlated negatively with S2, showing that PHT clearance diminishes with dose as it has been previously reported. Since a significant difference was found for S1/S2 ratio between male and females, CL is the pharmacokinetic parameter influenced by gender in PHT disposition.

Conclusion: S1/S2 saliva drug concentration ratio was sensitive enough for detecting systemic clearance changes. Both CBZ and PHT would modify their bioavailability and clearance by inducing efflux transporter throughout chronic treatments, from the first dose to multiple dose administration.
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http://dx.doi.org/10.2174/13816128113199990368DOI Listing
August 2014

P-glycoprotein contributes to cell membrane depolarization of hippocampus and neocortex in a model of repetitive seizures induced by pentylenetetrazole in rats.

Curr Pharm Des 2013 ;19(38):6732-8

Av. Caseros 1944 #9B, CABA (1152) - Argentina.

P-glycoprotein (P-gp) has been associated with pharmacoresistance and mechanisms regulating the membrane potential. However, at present it is unknown if P-gp overexpression in brain is associated with changes in membrane depolarization in refractory epilepsy. Experiments were designed to evaluate the membrane depolarization and P-gp overexpression induced by repetitive pentilenetetrazole (PTZ)-induced-seizures. Wistar rats were daily treated with PTZ during 4 to 7 days (PTZ4 and PTZ7 groups), and the brain was used to evaluate membrane potential by in vitro electrophysiological procedures and using bis-oxonol dye, [bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)], a fluorescence dye voltage-sensitive to membrane potentials. Rats with repetitive PTZ-induced seizures demonstrated lower phenytoin-induced anticonvulsant effects, increased number of DiBAC4(3) fluorescence cells and P-gp overexpression in hippocampus and neocortex, as well as augmentation of the induced fEPSP in CA1 field. These changes were more evident in PTZ7 group. Phenytoin or phenytoin plus nimodipine (a P-gp antagonist) avoided the enhanced fEPSP and decreased DiBAC4(3) fluorescence in animals from PTZ4 group. However, in PTZ7 group these effects were evident only when phenytoin was combined with nimodipine. An additional flow cytometry study demonstrated increased intracellular accumulation of DiBAC4(3) in K562 leukemic cells that overexpress MDR-1 and COX-2 genes, and are refractory to specific cytotoxic agents. These results represent the first evidence supporting the notion that brain P-gp overexpression contributes to a progressive seizure-related membranes depolarization in hippocampus and neocortex. Further experiments should be carried out to confirm the role of P-gp on membrane depolarization and epileptogenesis process.
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http://dx.doi.org/10.2174/1381612811319380006DOI Listing
August 2014
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