Ciprofloxacin permeability and its active secretion through rat small intestine in vitro

The biopharmaceutical aspect of the fluoroquinolones-metal cations interaction, which reduces antibacterial agents bioavailability and the mechanism of the fluoroquinolone intestinal efflux are still poorly understood. The purpose of this work was to gain further insights into these two issues by measuring the permeability of ciprofloxacin through the rat small intestine in side-by-side diffusion chambers using different incubation media and transport inhibitors. The permeability of ciprofloxacin from the mucosal to the serosal side was low. It was not influenced by the different concentrations of Ca(2+) and Mg(2+) in the donor solution. The active efflux of ciprofloxacin was the highest in the region of the rat small intestine excised proximal to the ileo-caecal junction or when the pH value of the incubation saline was slightly acidic. Thus ciprofloxacin appears to be transported in its cationic or in its zwitterionic form. The efflux was not inhibited by verapamil, benzbromarone or quinidine, which were added to the mucosal side of the intestinal tissue. It was however inhibited by quinidine added to the serosal side. The active secretion is therefore most probably a consequence of the organic cation transporter 1 activity at the basolateral membrane of enterocytes.     

The effect of clodronate on the integrity and viability of rat small intestine in vitro-a comparison with EDTA

Although substances, which can increase the paracellular permeability of intestinal mucosa, could be very helpful for increasing the bioavailability of hydrophilic drugs, they are not used therapeutically due to the possibilities of acute or long-term toxicity (intestinal inflammations due to penetration of bacterial fragments into subepithelial spaces). In this paper the abilities of a calcium chelator EDTA and clodronate (a first generation bisphosphonate) to increase the paracellular permeability were assessed using rat jejunum in side-by-side diffusion chambers while the viability of the tissue was monitored by transepithelial potential difference. Although clodronate is less potent than EDTA in depleting calcium from the intestinal tissue, it significantly increased the paracellular permeability of viable rat jejunum "in vitro" when tested at 15 mM and higher concentrations (the highest therapeutic dose dissolved in 250 ml gives a 22 mM solution of clodronate). This effect was reversible under "high-calcium" conditions. Since clodronate therapy does not have any long-term consequences it was concluded that a safe, transient increase of small intestinal permeability is possible. However, the acute gastrointestinal undesired effects, which can develop during the therapy with high doses of clodronate, might also occur after oral applications of paracellular permeability enhancers. Namely, 30 mM and higher concentrations of clodronate caused a loss of the tissue viability in all rat jejunal segments tested in "in vitro" conditions. A similar effect was observed with much lower concentrations of EDTA.     

Long-term amygdalohippocampal stimulation for refractory temporal lobe epilepsy

Short-term deep brain stimulation (DBS) recently has been shown to be efficacious in refractory temporal lobe epilepsy. We (1) evaluated long-term DBS in medial temporal lobe structures in patients with normal magnetic resonance imaging (MRI) findings and (2) investigated the use of chronic DBS electrodes for the localization of the ictal onset zone before DBS. In three patients with complex partial seizures (CPSs), DBS electrodes were implanted in the amygdalohippocampal region to identify and subsequently stimulate the ictal onset zone. CPSs were compared before and after chronic DBS. Side effects were carefully monitored. DBS electrodes yielded high-quality electroencephalogram recordings showing unilateral seizure onset in medial temporal lobe structures. For all patients, unilateral amygdalohippocampal stimulation was performed. After a mean follow-up of 5 months (range, 3-6 months), all patients had a greater than 50% reduction in seizure frequency. In two patients, antiepileptic drugs could be tapered. None of the patients reported side effects. This open study demonstrates the feasibility of consecutive electroencephalographic recordings and DBS in medial temporal lobe structures using DBS electrodes. These results prompt further studies in a larger patient population to establish the efficacy and safety of chronic DBS as an alternative treatment for refractory temporal lobe epilepsy.     

Direct medical costs of refractory epilepsy incurred by three different treatment modalities: a prospective assessment

PURPOSE: More than 20% of epilepsy patients have refractory seizures. Treatment options for these patients include continued polytherapy with/without novel antiepileptic drugs (AEDs), epilepsy surgery (ES), or vagus nerve stimulation (VNS). The purpose of this study was prospectively to compare epilepsy-related direct medical costs (ERDMCs) incurred by these different treatment modalities. METHODS: Eighty-four patients underwent a complete presurgical evaluation protocol at our institution. As a result, 24 (29%) patients were treated with continued AED polytherapy only; 35 (40%) underwent ES; and 25 (30%) had VNS. In each patient, annual costs in the 2 years preceding the therapeutic decision (ERDMC-pre) and during the follow-up afterward (ERDMC-post) were prospectively calculated. Furthermore, frequency of complex partial seizures with/without secondary generalization (CPS+/-SG), dosage and number of AEDs, number of hospital admission days, clinic visits, and laboratory tests before and after the therapeutic decision also were prospectively assessed. ERDMC-pre and ERDMC-post were compared in and among the three treatment groups. RESULTS: In patients conservatively treated with AEDs, mean frequency of CPSs decreased from 12 per month to nine per month, whereas mean ERDMCs decreased from $2,525 U.S. to $2,421 U.S. In surgical patients, mean seizure frequency decreased from six to fewer than one per month; mean ERDMCs per year decreased from $1,465 U.S. preoperatively to $1,186 U.S. postoperatively. In the VNS group, mean seizure frequency decreased from 21 per month to seven per month. ERDMCs in this subgroup decreased from $4,826 U.S. to $2,496 U.S. Mean seizure frequency changes were significant when conservatively treated patients were compared with surgically treated and VNS patient groups (chi2 test, p<0.001 and p=0.0019, respectively). ERDMC changes in conservatively treated patients also were statistically significant when compared with surgically treated and VNS patients (chi2 test, p=0.0007 and p=0.0036, respectively). No statistically significant differences were found in ERDMC changes between the surgical and VNS groups (chi2 test, p=0.387). CONCLUSIONS: Ongoing daily treatment of patients who underwent resective surgery costs significantly less than conservative treatment. For patients in whom resective surgery is not an option, ERDMC show a significant decrease in VNS-treated patients compared with conservatively treated patients.     

Polarised transport of monocarboxylic acid type drugs across rat jejunum in vitro: the effect of mucolysis and ATP-depletion

The transport characteristics of monocarboxylic acid type drugs (ketoprofen, ibuprofen and gemfibrozil) across the excised jejunal segments and artificial (octanol impregnated) membrane in side-by-side diffusion cells were studied. All three model drugs permeated faster across the intestinal tissue in the mucosal-to-serosal direction than in the opposite direction. No polarised transport of tested drugs was observed when the mucosal side of the intestine was treated with mucus disrupting agent, L-cysteine 1% (w/v), which significantly increased the microclimate pH at the mucosal surface of the intestine. Similar effects on the transport characteristics of model drugs and microclimate pH were observed when metabolic inhibitor, sodium azide (10mM), was present in the incubation medium. Furthermore, the direction of proton gradient across the artificial membrane was shown to significantly influence the transport of model drugs across this membrane. The results of this study indicate that the inwardly directed proton gradient maintained by the acidic microclimate pH at the intestinal surface could be considered as a driving force for the transport of monocarboxylic acid type drugs across the intestinal epithelia and could explain rapid absorption of these drugs after oral application.     

Effect of colloidal carriers on ascorbyl palmitate stability.

Active compounds can be protected against degradation by incorporation into colloidal carrier systems. The stabilizing effect of carrier systems for ascorbyl palmitate (AP) was investigated using microemulsions (ME), liposomes and solid lipid nanoparticles (SLN). Analysis of chemical stability by HPLC showed that AP is most resistant against oxidation in non-hydrogenated soybean lecithin liposomes, followed by SLN, w/o and o/w ME, and hydrogenated soybean lecithin liposomes. The molecular environment of the AP-like nitroxide probe (C(16)-Tempo) in colloidal carriers was characterized using electron paramagnetic resonance (EPR) spectroscopy. We have found that the nitroxide groups are located in environments with different polarity and mobility. The hydrophilic part of AP is the reactive moiety, and high stability is obtained in systems in which this part is exposed to a less polar environment. Additionally, the determined accessibility of nitroxide groups to reduction correlated well with the chemical stability of AP. It is more deeply immersed in the interface when entrapped in a liquid-state carrier than when applied in gel-state particles. Encapsulation of AP in SLN core leads to greater stability. We conclude that the location of the sensitive group of the drug-molecule in a carrier system is crucial for its stability.     

Detection of focal epileptiform events in the EEG by spatio-temporal dipole clustering

OBJECTIVE: Methods for the detection of epileptiform events can be broadly divided into two main categories: temporal detection methods that exploit the EEG's temporal characteristics, and spatial detection methods that base detection on the results of an implicit or explicit source analysis. We describe how the framework of a spatial detection method was extended to improve its performance by including temporal information. This results in a method that provides (i) automated localization of an epileptogenic focus and (ii) detection of focal epileptiform events in an EEG recording. For the detection, only one threshold value needs to be set. METHODS: The method comprises five consecutive steps: (1) dipole source analysis in a moving window, (2) automatic selection of focal brain activity, (3) dipole clustering to arrive at the identification of the epileptiform cluster, (4) derivation of a spatio-temporal template of the epileptiform activity, and (5) template matching. Routine EEG recordings from eight paediatric patients with focal epilepsy were labelled independently by two experts. The method was evaluated in terms of (i) ability to identify the epileptic focus, (ii) validity of the derived template, and (iii) detection performance. The clustering performance was evaluated using a leave-one-out cross validation. Detection performance was evaluated using Precision-Recall curves and compared to the performance of two temporal (mimetic and wavelet based) and one spatial (dipole analysis based) detection methods. RESULTS: The method succeeded in identifying the epileptogenic focus in seven of the eight recordings. For these recordings, the mean distance between the epileptic focus estimated by the method and the region indicated by the labelling of the experts was 8mm. Except for two EEG recordings where the dipole clustering step failed, the derived template corresponded to the epileptiform activity marked by the experts. Over the eight EEGs, the method showed a mean sensitivity and selectivity of 92 and 77%, respectively. CONCLUSIONS: The method allows automated localization of the epileptogenic focus and shows good agreement with the region indicated by the labelling of the experts. If the dipole clustering step is successful, the method allows a detection of the focal epileptiform events, and gave a detection performance comparable or better to that of the other methods. SIGNIFICANCE: The identification and quantification of epileptiform events is of considerable importance in the diagnosis of epilepsy. Our method allows the automatic identification of the epileptic focus, which is of value in epilepsy surgery. The method can also be used as an offline exploration tool for focal EEG activity, displaying the dipole clusters and corresponding time series.     

Vagus nerve stimulation for refractory epilepsy: a Belgian multicenter study.

INTRODUCTION: Vagus nerve stimulation (VNS) is a symptomatic add-on treatment for patients with medically refractory epilepsy. It consists of continuous electrical stimulation of the left vagus nerve by means of a helical electrode and an implantable, programmable pulse generator. Currently, over 50,000 patients are treated with VNS worldwide. AIM: This uncontrolled, open-label retrospective study evaluates long-term outcome in patients treated with VNS for refractory epilepsy in seven different epilepsy centres in Belgium. METHODS: For the purpose of this study, a minimum of essential inclusion criteria were defined to collect relevant data. This limited the results to basic findings with regards to efficacy on the long term. Inclusion criteria were a follow-up of at least 12 months and a documented seizure diary before implantation and at maximum follow-up. Primary outcome measures were the reduction in mean monthly seizure frequency and the percentage of patients with a seizure reduction of at least 50% (responder rate). RESULTS: About 138 patients (67M/71F) had a mean age of 30 years (range 4-59) at time of implantation and a mean post-implantation follow-up of 44 months (range 12-120). The mean number of AEDs before implantation was 3 (range 1-5). About 117/138 patients had focal epilepsy, 21 patients had symptomatic generalised epilepsy. About 117/138 patients were older than 16 years, 21 patients were 16 or younger. At maximum follow-up, mean stimulation output current was 1.84mA (range 0-3.25). Mean number of AEDs at maximum follow-up remained unchanged. The overall reduction in mean monthly seizure frequency was 51%. Mean seizure frequency before implantation was 41 seizures/month (SD=61; range 1-300), mean seizure frequency after implantation at maximum follow-up was 7 seizures/month (SD=25; range 0-120). Responder rate was 59%. 13% of patients had a seizure frequency decrease between 30% and 50%. About 28% had a seizure frequency decrease of<30%. Seizure freedom was obtained in 12/138 patients (9%). CONCLUSIONS: The long-term experience with VNS in Belgium confirms that VNS is an efficacious adjunctive antiepileptic treatment for patients with refractory epilepsy.     

A novel gene delivery system for stable transfection of thiopurine-S-methyltransferase gene in versatile cell types

A novel gene delivery system termed artificial viral particles (AVPs) containing a plasmid coding for a recombinant fusion protein of enhanced green fluorescent protein (EGFP) with thiopurine-S-methyltransferase (TPMT) was designed for transfection of selected cell lines to establish stable clones which express recombinant EGFP–TPMT protein for further in vitro investigation of toxic effect of thiopurine drugs. Various AVPs based on a complex of the cationic polymer polyethylenimine (PEI) and anionic liposomes were formulated and transfection conditions were adapted in order to transfect the human Jurkat, HepG2 and HEK 293 cell lines. An adequate transfection rate was achieved with AVP containing branched low molecular weight PEI at a PEI:DNA charge ratio of 4.5:1 and liposomes composed of DOPS, DLPE, cholesterol and an activated N-glutaryl-DOPE membrane anchor. Stably transfected clones were successfully established and expression of recombinant EGFP–TPMT in homogeneous cell populations was demonstrated by flow cytometry, fluorescence microscopy and immunoblotting. The level of the expressed protein in stable clones was highest in HEK 293, followed by HepG2 and Jurkat. The enzymatic activity of the TPMT moiety was demonstrated by decreased sensitivity to 6-thioguanine and increased sensitivity to 6-mercaptopurine in HEK 293 cells expressing EGFP–TPMT. Formulation of AVP as transfection vector succeeded in establishing human cell lines stably expressing EGFP–TPMT, thereby proving a successful delivery system and providing an initial step to enable investigation of the role of the clinically important drug metabolizing enzyme TPMT.     

Acute single photon emission computed tomographic study of vagus nerve stimulation in refractory epilepsy

PURPOSE: Left-sided vagus nerve stimulation (VNS) is an efficacious treatment for patients with refractory epilepsy. The precise mechanism of action remains to be elucidated. Only limited data on VNS-induced changes in regional cerebral blood flow (rCBF) are available. The aim of this study was to investigate rCBF changes during initial VNS with single-photon emission computed tomography (SPECT). METHODS: In 12 patients (8 women, 4 men) with mean age of 32 years and mean duration of epilepsy of 19 years, VNS-induced rCBF changes were studied by means of a 99mTc-ethyl cysteinate dimer activation study with a single-day split-dose protocol before and immediately after initial stimulation. Images were acquired on a triple-head camera with fan-beam collimators and were reconstructed with scatter and attenuation correction. After coregistration to a standardized template, both a semiquantitative analysis using predefined volumes-of-interest (VOIs) as well as voxel-by-voxel analysis of the intrasubject activation were performed. During follow-up, efficacy of VNS in terms of seizure-frequency reduction was studied. RESULTS: The semiquantitative analysis, with reference to the total counts in all VOIs, revealed a significant decrease of activity in the left thalamus immediately after the initial stimulation train. These results agreed with voxel-by-voxel analysis. In our study ipsilateral thalamic hypoperfusion was the most significant finding. Mean frequency of complex partial seizures was reduced from 30 per month before implantation to six per month after implantation. CONCLUSIONS: VNS induces rCBF changes immediately after initial stimulation that can be studied with SPECT. VNS-induced changes in the thalamus may play an important role in suppression of seizures. However, no significant relation between the level of hypoperfusion and subsequent clinical efficacy was found.