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Kreimann M.,University of Greifswald | Brandt S.,University of Greifswald | Krauel K.,Institute For Immunologie Und Transfusionsmedizin | Block S.,University of Greifswald | And 4 more authors.
Blood | Year: 2014

The chemokine platelet factor 4 (PF4) undergoes conformational changes when complexing with polyanions. This can induce the antibody-mediated adverse drug effect of heparin-induced thrombocytopenia (HIT). Understanding why the endogenous protein PF4 becomes immunogenic when complexing with heparin is important for the development of other negatively charged drugs and may also hint toward more general mechanisms underlying the induction of autoantibodies to other proteins. By circular dichroism spectroscopy, atomic force microscopy, and isothermal titration calorimetry we characterized the interaction of PF4 with unfractionated heparin (UFH), its 16-, 8-, and 6-mer subfractions, low-molecular-weight heparin (LMWH), and the pentasaccharide fondaparinux. To bind anti-PF4/heparin antibodies, PF4/heparin complexes require (1) an increase in PF4 anti-parallel β-sheets exceeding ∼30%(achieved by UFH, LMWH, 16-, 8-, 6-mer), (2) formation of multimolecular complexes (UFH, 16-, 8-mer), and (3) energy (needed for a conformational change), which is released by binding of ≥11-mer heparins to PF4, but not by smaller heparins. These findings may help to synthesize safer heparins. Beyond PF4 and HIT, the methods applied in the current study may be relevant to unravel mechanisms making other endogenous proteins more vulnerable to undergo conformational changes with little energy requirement (eg, point mutations and post-translational modifications) and thereby predisposing them to become immunogenic. © 2014 by The American Society of Hematology.


Lu R.,Goethe University Frankfurt | Lukowski R.,Institute For Pharmazie | Sausbier M.,Institute For Pharmazie | Zhang D.D.,Goethe University Frankfurt | And 6 more authors.
Pain | Year: 2014

Large conductance calcium-activated potassium (BKCa) channels are important regulators of neuronal excitability. Although there is electrophysiological evidence for BKCa channel expression in sensory neurons, their in vivo functions in pain processing have not been fully defined. Using a specific antibody, we demonstrate here that BKCa channels are expressed in subpopulations of peptidergic and nonpeptidergic nociceptors. To test a functional association of BKCa channel activity in sensory neurons with particular pain modalities, we generated mice in which BK Ca channels are ablated specifically from sensory neurons and analyzed their behavior in various models of pain. Mutant mice showed increased nociceptive behavior in models of persistent inflammatory pain. However, their behavior in models of neuropathic or acute nociceptive pain was normal. Moreover, systemic administration of the BKCa channel opener, NS1619, inhibited persistent inflammatory pain. Our investigations provide in vivo evidence that BKCa channels expressed in sensory neurons exert inhibitory control on sensory input in inflammatory pain states. © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.


Haag S.F.,Institute For Pharmazie | Taskoparan B.,Charité - Medical University of Berlin | Bittl R.,Free University of Berlin | Teutloff C.,Free University of Berlin | And 6 more authors.
Skin Pharmacology and Physiology | Year: 2011

The detection of the antioxidative capacity of the skin is of great practical relevance since free radicals are involved in many skin damaging processes, including aging and inflammation. The nitroxide TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxyl) in combination with electron paramagnetic resonance spectroscopy was found suitable for measuring the antioxidative capacity since its reaction with reducing agents is considerably fast. Yet, in order to achieve longer measurement times, e.g. in inflammatory skin diseases, the stabilizing effect of an invasome (ultraflexible vesicle/liposome) suspension with TEMPO was investigated ex vivo on porcine skin and in vivo on human skin. Invasomes increased the measurement time ex vivo 2-fold and the reduction was significantly slowed down in vivo, which is due to membrane-associated and therefore protected TEMPO. Furthermore, TEMPO accumulation in the membrane phase as well as the decreasing polarity of the ultimate surroundings of TEMPO during skin penetration explains the stabilizing effect. Thus, an invasome suspension with TEMPO exhibits stabilizing effects ex vivo and in vivo. Copyright © 2011 S. Karger AG, Basel.


Darras F.H.,Institute For Pharmazie | Pockes S.,Institute For Pharmazie | Huang G.,University of Würzburg | Wehle S.,University of Würzburg | And 5 more authors.
ACS Chemical Neuroscience | Year: 2014

Combination of AChE inhibiting and histamine H3 receptor antagonizing properties in a single molecule might show synergistic effects to improve cognitive deficits in Alzheimer's disease, since both pharmacological actions are able to enhance cholinergic neurotransmission in the cortex. However, whereas AChE inhibitors prevent hydrolysis of acetylcholine also peripherally, histamine H3 antagonists will raise acetylcholine levels mostly in the brain due to predominant occurrence of the receptor in the central nervous system. In this work, we designed and synthesized two novel classes of tri- and tetracyclic nitrogen-bridgehead compounds acting as dual AChE inhibitors and histamine H3 antagonists by combining the nitrogen-bridgehead moiety of novel AChE inhibitors with a second N-basic fragment based on the piperidinylpropoxy pharmacophore with different spacer lengths. Intensive structure-activity relationships (SARs) with regard to both biological targets led to compound 41 which showed balanced affinities as hAChE inhibitor with IC50 = 33.9 nM, and hH3R antagonism with Ki = 76.2 nM with greater than 200-fold selectivity over the other histamine receptor subtypes. Molecular docking studies were performed to explain the potent AChE inhibition of the target compounds and molecular dynamics studies to explain high affinity at the hH3R. © 2014 American Chemical Society.


Chen X.,Institute For Pharmazie | Chen X.,University of Würzburg | Wehle S.,University of Würzburg | Kuzmanovic N.,University of Regensburg | And 6 more authors.
ACS Chemical Neuroscience | Year: 2014

Photochromic cholinesterase inhibitors were obtained from cis-1,2-α-dithienylethene-based compounds by incorporating one or two aminopolymethylene tacrine groups. All target compounds are potent acetyl- (AChE) and butyrylcholinesterase (BChE) inhibitors in the nanomolar concentration range. Compound 11b bearing an octylene linker exhibited interactions with both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Yet upon irradiation with light, the mechanism of interaction varied from one photochromic form to another, which was investigated by kinetic studies and proved "photoswitchable". The AChE-induced β-amyloid (Aβ) aggregation assay gave further experimental support to this finding: Aβ1-40 aggregation catalyzed by the PAS of AChE might be inhibited by compound 11b in a concentration-dependent manner and seems to occur only with one photochromic form. Computational docking studies provided potential binding modes of the compound. Docking studies and molecular dynamics (MD) simulations for the ring-open and -closed form indicate a difference in binding. Although both forms can interact with the PAS, more stable interactions are observed for the ring-open form based upon stabilization of a water molecule network within the enzyme, whereas the ring-closed form lacks the required conformational flexibility for an analogous binding mode. The photoswitchable inhibitor identified might serve as valuable molecular tool to investigate the different biological properties of AChE as well as its role in pathogenesis of AD in in vitro assays. © 2014 American Chemical Society.


Lu R.,Witten/Herdecke University | Lu R.,Universitatsklinikum Frankfurt | Bausch A.E.,Institute For Pharmazie | Kallenborn-Gerhardt W.,Universitatsklinikum Frankfurt | And 9 more authors.
Journal of Neuroscience | Year: 2015

Slack (Slo2.2) is a sodium-activated potassium channel that regulates neuronal firing activities and patterns. Previous studies identified Slack in sensory neurons, but its contribution to acute and chronic pain in vivo remains elusive. Here we generated global and sensory neuron-specific Slack mutant mice and analyzed their behavior in various animal models of pain. Global ablation of Slack led to increased hypersensitivity in models of neuropathic pain, whereas the behavior in models of inflammatory and acute nociceptive pain was normal. Neuropathic pain behaviors were also exaggerated after ablation of Slack selectively in sensory neurons. Notably, the Slack opener loxapine ameliorated persisting neuropathic pain behaviors. In conclusion, Slack selectively controls the sensory input in neuropathic pain states, suggesting that modulating its activity might represent a novel strategy for management of neuropathic pain. © 2015 the authors.


Kaess K.,Institute For Pharmazie | Fahr A.,Institute For Pharmazie
European Journal of Lipid Science and Technology | Year: 2014

The effect of plasma on the retention of lipophilic drugs by liposomes as affected by the liposome fluidity was investigated. Liposomes consisted of eggPC/eggPG (9:1 w/w) or DPPC/DPPG (9:1 w/w) with 10 wt% incorporated 14C-labeled Temoporfin (mTHPC) and trace amounts of 3H-labeled cholesteryloleylether (COE) as lipid marker. Liposomal formulations were incubated at different concentrations in 50% human plasma for 48 h and the mixtures fractionated using gel permeation chromatography, separating liposomes and (lipo)proteins (albumin, HDL, LDL) from each other. The DPPC/DPPG liposomes retained 75-90% of the COE label at 48 h, depending on the liposome concentration, while the EPC/EPG liposomes retained only 5% of this label. By contrast, for the case of the mTHPC label, the EPC/EPG liposomes retained 24% at 48 h. whereas the DPPC/DPPG liposomes retained only 15%. In all cases, HDL was the predominant acceptor of both drug and COE label, closely followed by LDL. Albumin participated only marginally in the transfer process. It can be concluded, that EPC/EPG liposomes can retain lipophilic drugs better than DPPC/DPPG liposomes. Given a lifetime of 8 h for liposomes in vivo, 40% of the drug stays in the liposomes, whereas only 25% is retained for DPPC/DPPG liposomes at sufficient liposome concentration. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Siegert F.,Institute For Pharmazie | Nieber K.,Institute For Pharmazie
Medizinische Monatsschrift fur Pharmazeuten | Year: 2010

Irritable bowel syndrome is a common functional disorder of the gut. The cause is not known. Symptoms can be quite variable and include abdominal pain, bloating, and sometimes bouts of diarrhea and/or constipation. It causes a great deal of discomfort and distress, but it does not permanently harm the intestine and does not lead to a serious disease, such as cancer. There are numerous treatment options in functional gastrointestinal disorders acting peripherally by influencing motility and visceral sensitivity. However, older 5-HT 4 receptor agonists had limited clinical success because they were associated with changes in the cardiac function. New generation 5-HT4 receptor agonists, 5-HT3 antagonists or partial antagonists are promising approaches to treat gastrointestinal dysmotility, particularly colonic diseases. A further new approach is the activation of chloride cannels within the gastrointestinal wall by the prostaglandin E metabolite lubiprostone. In patients with chronic constipation, lubiprostone produced a bowel movement, with sustained improvement in frequency as well as other constipation symptoms. Ongoing clinical trials suggest that linaclotide, a first-in-class, 14-amino acid peptide guanylate cyclase C (GC-C) receptor agonist and intestinal secretagogue is also an effective treatment for chronic constipation. The pharmacological profile suggests that orally administered linaclotide may be capable of improving the abdominal symptoms and bowel habits of patients suffering from of constipation-predominant irritable bowel syndrome and chronic constipation. Data are emerging, but the efficacy and safety profile of these agents in the treatment irritable bowel disease appears encouraging. Further randomized controlled trials are warranted. ©2003-2010 MMP, Medizinische Monatsschrift f̈r Pharmazeuten, Wissenschaftliche Verlagsgesellschaft Stuttgart und Deutscher Apotheker Verlag.


Michael S.,University of Leipzig | Warstat C.,University of Leipzig | Michel F.,University of Leipzig | Yan L.,University of Bonn | And 3 more authors.
Purinergic Signalling | Year: 2010

Adenosine can show anti-inflammatory as well as pro-inflammatory activities. The contribution of the specific adenosine receptor subtypes in various cells, tissues and organs is complex. In this study, we examined the effect of the adenosine A2A receptor agonist CGS 21680 and the A2BR antagonist PSB-1115 on acute inflammation induced experimentally by 2,4,6-trinitrobenzenesulfonic acid (TNBS) on rat ileum/jejunum preparations. Pre-incubation of the ileum/jejunum segments with TNBS for 30 min resulted in a concentration-dependent inhibition of acetylcholine (ACh)-induced contractions. Pharmacological activation of the A2AR with CGS 21680 (0.1-10 μM) pre-incubated simultaneously with TNBS (10 mM) prevented concentration-dependently the TNBS-induced inhibition of the ACh contractions. Stimulation of A2BR with the selective agonist BAY 60-6583 (10 μM) did neither result in an increase nor in a further decrease of ACh-induced contractions compared to the TNBS-induced inhibition. The simultaneous pre-incubation of the ileum/jejunum segments with TNBS (10 mM) and the selective A2BR antagonist PSB-1115 (100 μM) inhibited the contraction-decreasing effect of TNBS. The effects of the A2AR agonist and the A2BR antagonist were in the same range as the effect induced by 1 μM methotrexate. The combination of the A2AR agonist CGS 21680 and the A2BR antagonist PSB-1115 at subthreshold concentrations of both agents found a significant amelioration of the TNBS-diminished contractility. Our results demonstrate that the activation of A2A receptors or the blockade of the A2B receptors can prevent the inflammation-induced disturbance of the ACh-induced contraction in TNBS pre-treated small intestinal preparations. The combination of both may be useful for the treatment of inflammatory bowel diseases. © 2009 Springer Science+Business Media B.V.


PubMed | Yale University and Institute For Pharmazie
Type: Journal Article | Journal: Learning & memory (Cold Spring Harbor, N.Y.) | Year: 2015

Kcnt1 encoded sodium-activated potassium channels (Slack channels) are highly expressed throughout the brain where they modulate the firing patterns and general excitability of many types of neurons. Increasing evidence suggests that Slack channels may be important for higher brain functions such as cognition and normal intellectual development. In particular, recent findings have shown that human Slack mutations produce very severe intellectual disability and that Slack channels interact directly with the Fragile X mental retardation protein (FMRP), a protein that when missing or mutated results in Fragile X syndrome (FXS), the most common form of inherited intellectual disability and autism in humans. We have now analyzed a recently developed Kcnt1 null mouse model in several behavioral tasks to assess which aspects of memory and learning are dependent on Slack. We demonstrate that Slack deficiency results in mildly altered general locomotor activity, but normal working memory, reference memory, as well as cerebellar control of motor functions. In contrast, we find that Slack channels are required for cognitive flexibility, including reversal learning processes and the ability to adapt quickly to unfamiliar situations and environments. Our data reveal that hippocampal-dependent spatial learning capabilities require the proper function of Slack channels.

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