Pharmacology and Drug Safety Research

Budapest, Hungary

Pharmacology and Drug Safety Research

Budapest, Hungary

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PubMed | Opto Neuropharmacology Group, University of Medicine and Pharmacy of Targu Mures, Hungarian Academy of Sciences, Pharmacology and Drug Safety Research and Semmelweis University
Type: | Journal: Frontiers in pharmacology | Year: 2015

The major drug binding site of sodium channels is inaccessible from the extracellular side, drug molecules can only access it either from the membrane phase, or from the intracellular aqueous phase. For this reason, ligand-membrane interactions are as important determinants of inhibitor properties, as ligand-protein interactions. One-way to probe this is to modify the pH of the extracellular fluid, which alters the ratio of charged vs. uncharged forms of some compounds, thereby changing their interaction with the membrane. In this electrophysiology study we used three different pH values: 6.0, 7.3, and 8.6 to test the significance of the protonation-deprotonation equilibrium in drug access and affinity. We investigated drugs of several different indications: carbamazepine, lamotrigine, phenytoin, lidocaine, bupivacaine, mexiletine, flecainide, ranolazine, riluzole, memantine, ritanserin, tolperisone, silperisone, ambroxol, haloperidol, chlorpromazine, clozapine, fluoxetine, sertraline, paroxetine, amitriptyline, imipramine, desipramine, maprotiline, nisoxetine, mianserin, mirtazapine, venlafaxine, nefazodone, and trazodone. We recorded the pH-dependence of potency, reversibility, as well as onset/offset kinetics. As expected, we observed a strong correlation between the acidic dissociation constant (pKa) of drugs and the pH-dependence of their potency. Unexpectedly, however, the pH-dependence of reversibility or kinetics showed diverse patterns, not simple correlation. Our data are best explained by a model where drug molecules can be trapped in at least two chemically different environments: A hydrophilic trap (which may be the aqueous cavity within the inner vestibule), which favors polar and less lipophilic compounds, and a lipophilic trap (which may be the membrane phase itself, and/or lipophilic binding sites on the channel). Rescue from the hydrophilic and lipophilic traps can be promoted by alkalic and acidic extracellular pH, respectively.


PubMed | Szent Istvan University, BioTalentum Ltd. and Pharmacology and Drug Safety Research
Type: Journal Article | Journal: Stem cell research | Year: 2016

Peripheral blood was collected from a clinically characterized female Kleefstra syndrome patient with a heterozygous, de novo, premature termination codon (PTC) mutation (NM_024757.4(EHMT1):c.3413G>A; p.Trp1138Ter). Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the human OSKM transcription factors using the Sendai-virus (SeV) delivery system. The pluripotency of transgene-free iPSC line was verified by the expression of pluripotency-associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. Furthermore, the iPSC line showed normal karyotype. Our model might offer a good platform to study the pathomechanism of Kleefstra syndrome, also for drug testing, early biomarker discovery and gene therapy studies.


Balogh P.,Semmelweis University | Magyar M.,Semmelweis University | Szabo A.,Semmelweis University | Mullner N.,Semmelweis University | And 3 more authors.
European Journal of Cell Biology | Year: 2015

We previously showed that intraperitoneal administration of Freund's adjuvant treatment resulted in acute peritonitis and TGF-β was found to be one of the main organizers of the subsequent EMT in mesothelial cells. In the present study, we investigated whether TGF-β signaling molecules are present in mesothelial cells and how their compartmentalization pattern changes with the dynamics of inflammatory events in vivo. In addition, we tried to evaluate the turnover of endosomal compartments concomitant with the internalization of signaling molecules and examine whether caveola-mediated internalization might play a role in the termination of TGF-β signaling. Using immunocytochemical approach, we could detect TβRII in EEA1 positive compartments and as the inflammation progressed, at D3, the receptor appeared in caveolin-1 positive intracellular structures as well. The latter event was accompanied by the appearance of negative regulatory protein, Smad7 in caveolae. We also found EEA1 and caveolin-1 double positive vesicular structures that were corresponded to forming MVBs affirmed by our immuno-electron microscopical results. Fine structural, morphometric and immunoblot analysis proved that Cd63 positive multivesicular body (MVB) formation was significantly increased by D3 and the IP results confirmed that TβRII as well as caveolin-1 were strongly associated with these endosomal compartments at this time. In contrast, by the termination of inflammation, by D5, caveolin-1 was found to be associated with late endosomal marker, Rab7 and entirely degraded from the system. Despite the limitations of an in vivo system, our results provide both morphological and biochemical data about the endosomal compartments involved in the internalization of TβRII upon inflammatory stimuli. Furthermore, our study implies the possible role of caveola-mediated endocytosis in the attenuation of TGF-β signaling and highlight the significance of endosomal compartments via which caveolae might meet the classical endocytic pathway under in vivo inflammatory conditions. © 2015 Elsevier GmbH.


Balogh P.,Semmelweis University | Magyar M.,Semmelweis University | Szabo A.,Semmelweis University | Mullner N.,Semmelweis University | And 3 more authors.
European journal of cell biology | Year: 2015

We previously showed that intraperitoneal administration of Freund's adjuvant treatment resulted in acute peritonitis and TGF-β was found to be one of the main organizers of the subsequent EMT in mesothelial cells. In the present study, we investigated whether TGF-β signaling molecules are present in mesothelial cells and how their compartmentalization pattern changes with the dynamics of inflammatory events in vivo. In addition, we tried to evaluate the turnover of endosomal compartments concomitant with the internalization of signaling molecules and examine whether caveola-mediated internalization might play a role in the termination of TGF-β signaling. Using immunocytochemical approach, we could detect TβRII in EEA1 positive compartments and as the inflammation progressed, at D3, the receptor appeared in caveolin-1 positive intracellular structures as well. The latter event was accompanied by the appearance of negative regulatory protein, Smad7 in caveolae. We also found EEA1 and caveolin-1 double positive vesicular structures that were corresponded to forming MVBs affirmed by our immuno-electron microscopical results. Fine structural, morphometric and immunoblot analysis proved that Cd63 positive multivesicular body (MVB) formation was significantly increased by D3 and the IP results confirmed that TβRII as well as caveolin-1 were strongly associated with these endosomal compartments at this time. In contrast, by the termination of inflammation, by D5, caveolin-1 was found to be associated with late endosomal marker, Rab7 and entirely degraded from the system. Despite the limitations of an in vivo system, our results provide both morphological and biochemical data about the endosomal compartments involved in the internalization of TβRII upon inflammatory stimuli. Furthermore, our study implies the possible role of caveola-mediated endocytosis in the attenuation of TGF-β signaling and highlight the significance of endosomal compartments via which caveolae might meet the classical endocytic pathway under in vivo inflammatory conditions. Copyright © 2015 Elsevier GmbH. All rights reserved.


PubMed | HAS SE Lendulet Hereditary Endocrine Tumors Research Group, Pharmacology and Drug Safety Research and Semmelweis University
Type: Journal Article | Journal: European journal of cell biology | Year: 2015

We previously showed that intraperitoneal administration of Freunds adjuvant treatment resulted in acute peritonitis and TGF- was found to be one of the main organizers of the subsequent EMT in mesothelial cells. In the present study, we investigated whether TGF- signaling molecules are present in mesothelial cells and how their compartmentalization pattern changes with the dynamics of inflammatory events in vivo. In addition, we tried to evaluate the turnover of endosomal compartments concomitant with the internalization of signaling molecules and examine whether caveola-mediated internalization might play a role in the termination of TGF- signaling. Using immunocytochemical approach, we could detect TRII in EEA1 positive compartments and as the inflammation progressed, at D3, the receptor appeared in caveolin-1 positive intracellular structures as well. The latter event was accompanied by the appearance of negative regulatory protein, Smad7 in caveolae. We also found EEA1 and caveolin-1 double positive vesicular structures that were corresponded to forming MVBs affirmed by our immuno-electron microscopical results. Fine structural, morphometric and immunoblot analysis proved that Cd63 positive multivesicular body (MVB) formation was significantly increased by D3 and the IP results confirmed that TRII as well as caveolin-1 were strongly associated with these endosomal compartments at this time. In contrast, by the termination of inflammation, by D5, caveolin-1 was found to be associated with late endosomal marker, Rab7 and entirely degraded from the system. Despite the limitations of an in vivo system, our results provide both morphological and biochemical data about the endosomal compartments involved in the internalization of TRII upon inflammatory stimuli. Furthermore, our study implies the possible role of caveola-mediated endocytosis in the attenuation of TGF- signaling and highlight the significance of endosomal compartments via which caveolae might meet the classical endocytic pathway under in vivo inflammatory conditions.


Lenkey N.,Hungarian Academy of Sciences | Karoly R.,Hungarian Academy of Sciences | Lukacs P.,Hungarian Academy of Sciences | Vizi E.S.,Hungarian Academy of Sciences | And 3 more authors.
PLoS ONE | Year: 2010

Background: There is only one established drug binding site on sodium channels. However, drug binding of sodium channels shows extreme promiscuity: ~25% of investigated drugs have been found to potently inhibit sodium channels. The structural diversity of these molecules suggests that they may not share the binding site, and/or the mode of action. Our goal was to attempt classification of sodium channel inhibitors by measuring multiple properties of inhibition in electrophysiology experiments. We also aimed to investigate if different properties of inhibition correlate with specific chemical properties of the compounds. Methodology/Principal Findings: A comparative electrophysiological study of 35 compounds, including classic sodium channel inhibitors (anticonvulsants, antiarrhythmics and local anesthetics), as well as antidepressants, antipsychotics and neuroprotective agents, was carried out using rNav1.2 expressing HEK-293 cells and the QPatch automatic patch-clamp instrument. In the multi-dimensional space defined by the eight properties of inhibition (resting and inactivated affinity, potency, reversibility, time constants of onset and offset, use-dependence and state-dependence), at least three distinct types of inhibition could be identified; these probably reflect distinct modes of action. The compounds were clustered similarly in the multi-dimensional space defined by relevant chemical properties, including measures of lipophilicity, aromaticity, molecular size, polarity and electric charge. Drugs of the same therapeutic indication typically belonged to the same type. We identified chemical properties, which were important in determining specific properties of inhibition. State-dependence correlated with lipophilicity, the ratio of the neutral form of molecules, and aromaticity: We noticed that the highly state dependent inhibitors had at least two aromatic rings, logP>4.0, and pKa<8.0. Conclusions/Significance: The correlations of inhibition properties both with chemical properties and therapeutic profiles would not have been evident through the sole determination of IC50; therefore, recording multiple properties of inhibition may allow improved prediction of therapeutic usefulness. © 2010 Lenkey et al.


Balogh P.,Semmelweis University | Szabo A.,Semmelweis University | Katz S.,Semmelweis University | Liko I.,Pharmacology and Drug Safety Research | And 2 more authors.
PLoS ONE | Year: 2013

Transformation of epithelial cells into connective tissue cells (epithelial-mesenchymal transition, EMT) is a complex mechanism involved in tumor metastasis, and in normal embryogenesis, while type II EMT is mainly associated with inflammatory events and tissue regenaration. In this study we examined type II EMT at the ultrastructural and molecular level during the inflammatory process induced by Freund's adjuvant treatment in rat mesenteric mesothelial cells. We found that upon the inflammatory stimulus mesothelial cells lost contact with the basal lamina and with each other, and were transformed into spindle-shaped cells. These morphological changes were accompanied by release of interleukins IL-1alpha, -1beta and IL-6 and by secretion of transforming growth factor beta (TGF-β) into the peritoneal cavity. Mesothelial cells also expressed estrogen receptor alpha (ER-α) as shown by immunolabeling at the light and electron microscopical levels, as well as by quantitative RT-PCR. The mRNA level of ER-α showed an inverse correlation with the secretion of TGF-β. At the cellular and subcellular levels ER-α was colocalized with the coat protein caveolin-1 and was found in the plasma membrane of mesothelial cells, in caveolae close to multivesicular bodies (MVBs) or in the membrane of these organelles, suggesting that ER-α is internalized via caveola-mediated endocytosis during inflammation. We found asymmetric, thickened, electron dense areas on the limiting membrane of MVBs (MVB plaques) indicating that these sites may serve as platforms for collecting and organizing regulatory proteins. Our morphological observations and biochemical data can contribute to form a potential model whereby ER-α and its caveola-mediated endocytosis might play role in TGF-β induced type II EMT in vivo. © 2013 Balogh et al.


Gyertyan I.,Pharmacology and Drug Safety Research | Kiss B.,Pharmacology and Drug Safety Research | Saghy K.,Pharmacology and Drug Safety Research | Laszy J.,Pharmacology and Drug Safety Research | And 10 more authors.
Neurochemistry International | Year: 2011

We investigated the in vivo effects of orally administered cariprazine (RGH-188; trans-N-{4-[2-[4-(2,3-dichlorophenyl)-piperazin-1-yl]-ethyl]- cyclohexyl}-N′,N′-dimethyl-urea), a D3/D2 dopamine receptor partial agonist with ∼10-fold preference for the D 3 receptor. Oral bioavailability of cariprazine at a dose of 1 mg/kg in rats was 52% with peak plasma concentrations of 91 ng/mL. Cariprazine 10 mg/kg had good blood-brain barrier penetration, with a brain/plasma AUC ratio of 7.6:1. In rats, cariprazine showed dose-dependent in vivo displacement of [3H](+)-PHNO, a dopamine D3 receptor-preferring radiotracer, in the D3 receptor-rich region of cerebellar lobules 9 and 10. Its potent inhibition of apomorphine-induced climbing in mice (ED 50 = 0.27 mg/kg) was sustained for 8 h. Cariprazine blocked amphetamine-induced hyperactivity (ED50 = 0.12 mg/kg) and conditioned avoidance response (CAR) (ED50 = 0.84 mg/kg) in rats, and inhibited the locomotor-stimulating effects of the noncompetitive NMDA antagonists MK-801 (ED50 = 0.049 mg/kg) and phencyclidine (ED50 = 0.09 mg/kg) in mice and rats, respectively. It reduced novelty-induced motor activity of mice (ED50 = 0.11 mg/kg) and rats (ED50 = 0.18 mg/kg) with a maximal effect of 70% in both species. Cariprazine produced no catalepsy in rats at up to 100-fold dose of its CAR inhibitory ED50 value. Cariprazine 0.02-0.08 mg/kg significantly improved the learning performance of scopolamine-treated rats in a water-labyrinth learning paradigm. Though risperidone, olanzapine, and aripiprazole showed antipsychotic-like activity in many of these assays, they were less active against phencyclidine and more cataleptogenic than cariprazine, and had no significant effect in the learning task. The distinct in vivo profile of cariprazine may be due to its higher affinity and in vivo binding to D3 receptors versus currently marketed typical and atypical antipsychotics. © 2011 Elsevier B.V. All rights reserved.


Tarnawa I.,Pharmacology and Drug Safety Research
Drugs of the Future | Year: 2012

Increased neuronal activity and/or excitability in particular regions of the peripheral or central nervous system is associated with a great variety of diseases, like chronic pain, epilepsy, neurodegenerative disorders and several psychiatric conditions. Abnormal overfunctioning of volt-age- gated sodium channels frequently underlies these disease states. Developing agents selectively targeting sodium channel subtypes involved in a particular disease is an attractive idea, since their use could be associated with a lower risk of side effects. Currently available drugs targeting sodium channels, however, are nonselective regarding channel subtypes and still have some preference towards diseased neurons because their actions are channel state dependent. They preferentially inhibit channels in the open or inactivated states, which are located abundantly on overactive cells. New results, reviewed in this paper, provide a deeper understanding of the role of sodium channels in the pathomechanism of neuropsychiatric disorders, as well as the interaction of sodium channel inhibitors with their binding sites on the channel. This knowledge, together with a considerable advancement in drug screening technologies, holds the promise that sodium channel inhibitors with improved therapeutic and side effect profiles can be developed. Copyright © 2012 Prous Science, S.A.U. or its licensors. All rights reserved.


PubMed | Pharmacology and Drug Safety Research
Type: | Journal: Brain research | Year: 2015

The trigeminovascular system has a pivotal role in the pathomechanism of migraine. The aim of the present study was to further develop existing models of migraine making them more suitable for testing the effects of compounds with presumed antimigraine activity in anaesthetised rats. Simultaneous recording of ongoing activity of spontaneously active neurons in the trigeminocervical complex as well as their discharges evoked by electrical stimulation of the dura mater via activation of A- and C-sensory fibres were carried out. Effects of sumatriptan, propranolol and topiramate were evaluated prior to and after application of a mixture containing inflammatory mediators on the dura. Propranolol (10 mg/kg s.c) and topiramate (30 mg/kg s.c.) resulted in a tendency to decrease the level of both spontaneous and evoked activity, while sumatriptan (1 mg/kg s.c.) did not exhibit any effect on recorded parameters. Application of an inflammatory soup to the dura mater boosted up spontaneous activity, which could be significantly attenuated by propranolol and topiramate but not by sumatriptan. In addition, all compounds prevented the delayed increase of spontaneous firing. In contrast to the ongoing activity, evoked responses were not augmented by inflammatory mediators. Nevertheless, inhibitory effect of propranolol and topiramate was evident when considering A- or C-fibre responses. Findings do not support the view that electrically evoked responses are useful for the measurement of trigeminal sensitization. It is proposed however, that inhibition of enhanced firing (immediate and/or delayed) evoked by inflammatory mediators as an endpoint have higher predictive validity regarding the clinical effectiveness of compounds.

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