The Effect of Cooling Fluid Composition on Ablation Size in Hepatic Laser Ablation: A Comparative Study in an Ex Vivo Bovine Setting

Purpose: Hyperthermic ablation is a minimally invasive mode of tumour therapy which serves as a viable alternative to surgical intervention. However, one of the major drawbacks, besides the heat sink effect and the risk of damaging adjacent organs, is limited ablation size. The use of a cooling fluid during ablation has been shown to increase the ablation volume and decrease the carbonisation rate. The aim of this study was to investigate whether the composition of the cooling fluid has an effect on ablation size and carbonisation rate during hepatic laser ablation in an ex vivo bovine setting. Method: In this study bovine hepatic tissue was ablated in an ex vivo setting using an internally cooled laser applicator. A total of 45 tissue samples were assigned to three groups: 0.9% saline infusion (n = 15), distilled water infusion (n = 15) and a 50%/50% mixture of 0.9% saline and distilled water (n = 15). Ablation was conducted using a 1064 nm Nd:YAG laser at a wattage of 25 W and time interval of 10 min. The ablation volume and carbonisation rate were then measured and recorded through postprocedural MRI. One-way ANOVA and post-hoc testing were performed to assess the effect of the cooling fluid composition on the ablation volumes. Results: We found that using a mixture of saline and distilled water as a cooling fluid during hyperthermic ablation resulted in a larger ablation volume (mean ± SD: 22.64 ± 0.99 cm3) when compared to saline infusion (21.08 ± 1.11 cm3) or distilled water infusion (20.92 ± 0.92 cm3). This difference was highly significant (p &lt; 0.001). There was no significant difference in ablation size between the saline group and the distilled water group. The highest carbonisation rate occurred in the saline group (12/15), followed by the mixed infusion group (3/15) and the distilled water group (1/15). Conclusions: The results of this study suggest that cooling fluid composition during hepatic laser ablation affects ablation volume in an ex vivo bovine setting. There was no statistically significant difference when comparing ablation volumes during saline infusion and distilled water infusion, but the carbonisation rate was significantly higher when using saline. The combination of saline and distilled water in a 50%/50% mixture as cooling fluid appears to be an auspicious alternative, as ablation volumes created with it are larger when compared to saline and distilled water alone, while carbonisation rate remains low. This might improve patient outcome as well as patient eligibility for hyperthermic ablation.</p

Comparison of In Vitro and In Vivo Results Using the GastroDuo and the Salivary Tracer Technique: Immediate Release Dosage Forms under Fasting Conditions

The fasted state administration of immediate release (IR) dosage forms is often regarded as uncritical since physiological aspects seem to play a minor role for disintegration and drug release. However, recent in vivo studies in humans have highlighted that fasted state conditions are in fact highly dynamic. It was therefore the aim of this study to investigate the disintegration and drug release behavior of four different IR formulations of the probe drug caffeine under physiologically relevant conditions with the aid of the GastroDuo. One film-coated tablet and three different capsule formulations based on capsule shells either made from hard gelatin or hydroxypropylmethyl cellulose (HPMC) were tested in six different test programs. To evaluate the relevance of the data generated, the four IR formulations were also studied in a four-way cross-over study in 14 healthy volunteers by using the salivary tracer technique (STT). It could be shown that the IR formulations behaved differently in the in vitro test programs. Thereby, the simulated parameters affected the disintegration and dissolution behavior of the four IR formulations in different ways. Whereas drug release from the tablet started early and was barely affected by temperature, pH or motility, the different capsule formulations showed a longer lag time and were sensitive to specific parameters. However, once drug release was initiated, it typically progressed with a higher rate for the capsules compared to the tablet. Interestingly, the results obtained with the STT were not always in line with the in vitro data. This observation was due to the fact that the probability of the different test programs was not equal and that certain scenarios were rather unlikely to occur under the controlled and standardized conditions of clinical studies. Nonetheless, the in vitro data are still valuable as they allowed to discriminate between different formulations

Die Funktion des MRP4 (ABCC4)-Transporters in Thrombozyten: Bedeutung von Adaptorproteinen

Das Multidrug Resistance Protein 4 (MRP4/ABCC4) ist als Mitglied der ABC-Transporterfamilie nicht nur an dem Transport zahlreicher Pharmaka, wie beispielsweise antiviraler und zytostatischer Substanzen, sondern auch an Signaltransduktionsprozessen, z.B. dem Transport von Eicosanoiden und zyklischen Nukleotiden beteiligt. MRP4 weist außerdem innerhalb der ABCC-Gruppe ein einmaliges Expression-, Lokalisations- und Substratspektrum auf. MRP4 wurde neben Prostata, Niere, Gehirn und Leber auch in Blutplättchen nachgewiesen und kann zelltypabhängig sowohl apikal oder basolateral als auch intrazellulär lokalisiert sein. Insbesondere das Vorkommen in den δ-Granula der Thrombozyten ist bemerkenswert, da die Speicherung und Freisetzung von Überträgersubstanzen wie ADP auf die Thrombozytenfunktion entscheidenden Einfluss haben. Änderungen der zelltypischen MRP4-Lokalisation, die beispielsweise bei Patienten mit δ-storage pool Defekt beobachtet wurden, können zu einem Verlust der spezifischen Transporterfunktion führen. Das Ziel dieser Arbeit war es, das Transportprotein Multidrug resistance protein 4 in Bezug auf Protein-Protein-Wechselwirkungen genauer zu untersuchen. Da die Lokalisation von Membranproteinen u.a. durch die Wechselwirkung mit Proteinen gesteuert wird, stand die Identifikation möglicher Interaktionspartner von MRP4 mit Hilfe von Bindungs- und Kolokalisationsstudien im Vordergrund dieser Arbeit. Es schien sehr wahrscheinlich, dass Adaptormoleküle über eine Wechselwirkung mit MRP4 an dessen trafficking innerhalb der Zellen beteiligt sind und damit Einfluss auf dessen Lokalisation und konsekutiv auch auf die Funktion nehmen. Als mögliche Motive zur Vermittlung solcher Proteinbindungen liegen im MRP4-Molekül ein PDZ-Motiv sowie eine mögliche Bindungsstelle für Adaptorprotein (AP)-Komplexe vor. Zur Ermittlung solcher potentiellen Partner wurde eine Affinitätschromatographie durchgeführt. Dafür erfolgte die Kopplung eines Peptids, welches der C-terminalen Sequenz von MRP4 mit dem PDZ-Bindemotiv entsprach, an eine Sepharosematrix und eine anschließende Inkubation mit Thrombozytenlysat. Mittels Western Blot-Verfahren und Flüssigkeitschromatographie-Massenspektrometrie konnten im gewonnenen Eluat das ERM-bindende Phosphoprotein 50 (EBP50/NHERF1), Moesin, sowie das Post synaptic density protein 95 (PSD95) und das Hitzeschockprotein Hsp90 als mögliche Bindungspartner identifiziert werden. Während eine Wechselwirkung von MRP4 mit EBP50 über seine PDZ-Domäne bereits postuliert worden war, konnten insbesondere PSD95 und Hsp90 erstmalig als mögliche Interaktionspartner von MRP4 ermittelt werden. Da PSD95 bisher vorwiegend in neuronalen Zellen beschrieben wurde, wurde das Vorkommen dieses Proteins in Thrombozyten und der megakaryoblastischen Leukämiezelllinie M-07e auf RNA-Ebene untersucht und nachgewiesen. Damit konnte erstmals die Expression dieses scaffolding Proteins in Zellen der myeloischen Reihe gezeigt werden. Im Anschluss daran wurden Kofärbungen von MRP4 und den identifizierten Bindungspartnern durchgeführt. Die indirekte Immunfluoreszenzmikroskopie lieferte das Ergebnis einer zumindest partiellen Kolokalisation des Transporters mit Hsp90 – in Thrombozyten und M-07e-Zellen in intrazellulären Strukturen, in den Nierenepithelzellen LLC-PK1 und MDCKII in der Plasmamembran. Auch eine Kolokalisation von MRP4 mit PSD95 konnte in M-07e-Zellen und Thrombozyten beobachtet werden. Untersuchungen mit dem Hsp90-Hemmstoff Radicicol ergaben des Weiteren zum einen eine sichtbare Verringerung der MRP4-Expression im Western Blot nach der Inkubation, zum anderen auch eine Änderung der Lokalisation von Hsp90 und MRP4 in den verwendeten Nierenzelllinien nach intrazellulär. Ferner konnten funktionelle Transportversuche unter Verwendung von inside-out Thrombozytenmembranvesikeln einen Abfall der Aufnahme des radioaktiv markierten MRP4-Substrats cGMP in die Vesikel nach Behandlung mit Radicicol zeigen. Um den Einfluss von PSD95 auf die MRP4-Lokalisation zu untersuchen, wurde ein spezifischer knock-down von PSD95 mittels siRNA in M-07e-Zellen durchgeführt. Im Anschluss ergab sich in der Immunfluoreszenzmikroskopie eine deutliche Zunahme der MRP4-Lokalisation in der Plasmamembran. Zusammenfassend konnten im Rahmen dieser Arbeit EBP50, Moesin, PSD95 und Hsp90 als mögliche Bindungspartner von MRP4 in Thrombozyten identifiziert werden. Es ist denkbar, dass Hsp90 als Hitzeschockprotein möglicherweise zu der Prozessierung und Stabilisierung von MRP4 beiträgt. Die Interaktion mit PSD95 hingegen scheint die Internalisierung des Transportproteins zu begünstigen. Die gewonnenen Ergebnisse deuten darauf hin, dass Protein-Protein-Interaktionen die Lokalisation und Funktion von MRP4 entscheidend beeinflussen.The Multidrug Resistance protein 4 (MRP4) is a member of the ABC-transporter family. It is not only involved in the transport of a number of drugs, such as antiviral and cytotoxic substances, but also in signal transduction processes, e.g. the transport of prostanoids and cyclic nucleotids. Among the C subgroup of ABC-transporters MRP4 possesses a uniquely broad pattern of expression, localisation and substrates transported. It was detected in prostate, kidney, brain, liver as well as in platelets and is expressed according to the specific cell type apically, basolaterally or even intracellulary. Especially its expression in platelets’ dense granules is noteworthy, as the storage and degranulation of mediators such as ADP are essential for platelet function. Changes in the location of MRP4, which can be seen in patients with δ-storage pool defects can lead to a loss of transporter function. The aim of this work was to examine the transport protein MRP4 concerning protein-protein interactions. As the localisation of membrane proteins is among others regulated by interaction with other proteins, the identification of possible partners by binding and colocalisation studies was the primary object of this study. It seemed likely, that adaptor molecules are involved in the trafficking of MRP4 and thereby influence its localization and consecutively its function. As possible binding motives MRP4 possesses a PDZ-motive and a possible interaction domain for adaptor protein complexes. To determine possible binding partners affinity chromatography was carried out. A short peptide of the MRP4 C-terminus was coupled to a sepharose matrix and afterwards incubated with platelet lysate. In the eluate ERM-binding phosphoprotein 50 (EBP50/NHERF1), Moesin, Post synaptic density protein 95 (PSD95) and Heat shock protein 90 (Hsp90) could be detected by using western blot and mass spectrometry analyses. A possible interaction of MRP4 with EBP50 has already been postulated. However, an interaction with PSD95 and Hsp90 was shown for the first time. Since PSD95 has until now only been described in neuronal structures, its expression in platelets and cells of megacaryoblastic origin was examined and proven on mRNA level. Afterwards, costaining of MRP4 and its possible binding partners was undertaken. Indirect immunofluorescence microscopy showed a colocalisation with Hsp90 in platelets and mecacaryoblastic cell line in intracellular compartments and in kidney derived cell lines in the plasma membrane. Likewise, a colocalisation of MRP4 and PSD95 could be seen in megacaryoblastic cells and platelets. Examinations using the Hsp90 inhibitor Radicicol showed a decrease in MRP4 expression in western bot analysis and a change in localisation of the transporter and Hsp90 in kidney cell lines to intracellular compartments. Moreover, a significant reduction of cGMP uptake into inside-out membrane vesicles of platelets could be detected after incubation with Radicicol in functional transport studies. To examine the influence of PSD95 on MRP4 localisation specific siRNA knock-down was carried out in a megacaryoblastic cell line. The following immunofluorescence revealed an increased expression of MRP4 in the plasma membrane. In conclusion, EBP50, Moesin, PSD95 and Hsp90 were detected as possible binding partners of MRP4 in platelets. It seems likely, that Hsp90 is involved in the processing and stabilization of MRP4. The interaction with PSD95 seems to lead to an internalization of the transport protein. The results point at an important influence of protein-protein interactions on the localization and function of MRP4

Pulmonary emphysema is a predictor of pneumothorax after CT-guided transthoracic pulmonary biopsies of pulmonary nodules.

Pneumothoraces are the most frequently occurring complications of CT-guided percutaneous transthoracic pulmonary biopsies (PTPB). The aim of this study was to evaluate the influence of pre-diagnostic lung emphysema on the incidence and extent of pneumothoraces and to establish a risk stratification for the evaluation of the pre-procedure complication probability.CT-guided PTPB of 100 pre-selected patients (mean age 67.1±12.8 years) were retrospectively enrolled from a single center database of 235 PTPB performed between 2012-2014. Patients were grouped according to pneumothorax appearance directly after PTPB (group I: without pneumothorax, n = 50; group II: with pneumothorax, n = 50). Group II was further divided according to post-interventional treatment (group IIa: chest tube placement, n = 24; group IIb: conservative therapy, n = 26). For each patient pre-diagnostic percentage of emphysema was quantified using CT density analysis. Emphysema stages were compared between groups using bivariate analyses and multinomial logistic regression analyses.Emphysema percentage was significantly associated with the occurrence of post-interventional pneumothorax (p = 0.006). Adjusted for potential confounders (age, gender, lesion size and length of interventional pathway) the study yielded an OR of 1.07 (p = 0.042). Absolute risk of pneumothorax increased from 43.4% at an emphysema rate of 5% to 73.8% at 25%. No differences could be seen in patients with pneumothorax between percentage of emphysema and mode of therapy (p = 0.721).The rate of lung emphysema is proportionally related to the incidence of pneumothorax after CT-guided PTPB and allows pre-interventional risk stratification. There is no association between stage of emphysema and post-interventional requirement of chest tube placement

Exploring factors associated with non-alcoholic fatty liver disease using longitudinal MRI

Abstract Background To identify factors associated with non-alcoholic fatty liver disease over a 5-year period. Methods Three hundred seven participants, including 165 women, with a mean age of 55.6 ± 12.0 years underwent continuous quantitative MRI of the liver using the proton-density fat fraction (PDFF). The liver’s fat fractions were determined at baseline and 5 years later, and the frequency of participants who developed fatty liver disease and potential influencing factors were explored. Based on significant factors, a model was generated to predict the development of fatty liver disease. Results After excluding participants with pre-existing fatty liver, the baseline PDFF of 3.1 ± 0.9% (n = 190) significantly increased to 7.67 ± 3.39% within 5 years (p < 0.001). At baseline, age (OR = 1.04, p = 0.006, CI = 1.01–1.07), BMI (OR = 1.11, p = 0.041, CI = 1.01–1.23), and waist circumference (OR = 1.05, p = 0.020, CI = 1.01–1.09) were identified as risk factors. Physical activity was negatively associated (OR = 0.43, p = 0.049, CI = 0.18–0.99). In the prediction model, age, physical activity, diabetes mellitus, diastolic blood pressure, and HDL-cholesterol remained as independent variables. Combining these risk factors to predict the development of fatty liver disease revealed an AUC of 0.7434. Conclusions Within a five-year follow-up, one-quarter of participants developed fatty liver disease influenced by the triggering factors of age, diabetes mellitus, low HDL-cholesterol, and diastolic blood pressure. Increased physical activity has a protective effect on the development of fatty liver

In Vivo Evaluation of a Gastro-Resistant Enprotect® Capsule under Postprandial Conditions

Ready-to-fill enteric hard capsule shells are an evolving field of oral drug and nutraceutical products. Lonza Capsugel® Enprotect® capsules were recently proven to provide reliable release in the small intestine after fasted intake, but robustness against postprandial intake needed to be proven. In this study, the capsules were administered to 16 healthy young subjects after intake of a light meal. The Enprotect® capsules were labelled with 5 mg black iron oxide and 25 mg 13C3-caffeine. Magnetic Resonance Imaging was used to identify the localization and visual dispersion of the capsule filling. The salivary appearance of caffeine was considered a second independent and sensitive marker for the initial release. Whereas the fasted gastric residence time of the capsules amounted to 43 ± 32 min, it was increased to 158 ± 36 min after postprandial intake. Therefore, the mean dispersion time according to MRI and the mean caffeine appearance time were increased to 196 ± 37 min and 189 ± 37 min, respectively. But, similar to fasted administration, no capsule disintegration or leakage was observed in the stomach and 38% of the capsules disintegrated in the jejunum and 62% in the ileum. The mean dispersion time after gastric emptying and the mean caffeine appearance time after gastric emptying amounted to 38 ± 21 min and 31 ± 17 min, respectively. Both did not relevantly change compared to the fasted intake. Only the absolute dispersion time and caffeine appearance were prolonged due to the increased gastric residence and no relevant influence of the light meal was observed on the disintegration or release behavior of Enprotect® capsules after gastric emptying. The capsules also showed robust enteric properties after postprandial administration

In Vivo Evaluation of a Gastro-Resistant HPMC-Based “Next Generation Enteric” Capsule

Many orally dosed APIs are bioavailable only when formulated as an enteric dosage form to protect them from the harsh environment of the stomach. However, an enteric formulation is often accompanied with a higher development effort in the first place and the potential degradation of fragile APIs during the coating process. Ready-to-use enteric hard capsules would be an easily available alternative to test and develop APIs in enteric formulations, while decreasing the time and cost of process development. In this regard, Lonza Capsugel® Next Generation Enteric capsules offer a promising approach as functional capsules. The in vivo performance of these capsules was observed with two independent techniques (MRI and caffeine in saliva) in eight human volunteers. No disintegration or content release in the stomach was observed, even after highly variable individual gastric residence times (range 7.5 to 82.5 min), indicating the reliable enteric properties of these capsules. Seven capsules disintegrated in the distal part of the small intestine; one capsule showed an uncommonly fast intestinal transit (15 min) and disintegrated in the colon. The results for this latter capsule by MRI and caffeine appearance differed dramatically, whereas for all other capsules disintegrating in the small intestine, the results were very comparable, which highlights the necessity for reliable and complementary measurement methods. No correlation could be found between the gastric residence time and disintegration after gastric emptying, which confirms the robust enteric formulation of those capsules