Modulation of the apical efflux transporter P-glycoprotein by Vitamin E TPGS: Structure-activity relationships and mechanism of inhibition

The non-ionic surfactant D-alpha-tocopheryl poly(ethylene glycol 1000) succinate (TPGS 1000) has been previously shown to increase oral bioavailability of P-glycoprotein (P-gp) substrates by modulating activity of the efflux pump. In the present thesis structure activity relationship of the interaction and possible mechanism of inhibition of the efflux transporter were investigated to optimize the TPGS structure. P-gp inhibitory activity of TPGS could be increased by modifying the length of the PEG chain with optimal inhibition being achieved at a PEG molecular weight of ~1500 Da. The inhibitory effect of TPGS on P-gp could be also increased by modifications to the hydrophobic part of the molecule, such as the exchange of the alpha-tocopherol moiety for cholesterol. The inhibitory activity of the TPGS analogues does not correlate with their physicochemical properties such as molecular weight, molecule volume, or lipophilicity. In agreement with these findings, an unspecific alteration of the P-gp membrane environment could be ruled out in electron spin resonance experiments. TPGS was shown not to be a P-gp substrate itself nor does it interact with one of the transport active drug binding sites of P-gp to competitively block drug efflux. Rather, the inhibitory effect of the TPGS analogues correlates with their inhibition of substrate induced ATPase activity, indicating that the depletion of the energy source of the efflux pump is an integral part in the inhibitory mechanism of TPGS.Für das nicht ionische Tensid D-Alpha-tocopheryl poly(ethylene glycol 1000) succinat (TPGS 1000) wurde kürzlich gezeigt, dass es die orale Bioverfügbarkeit von Substraten des Effluxtransporters P-Glykoprotein (P-gp) durch Hemmung der Effluxpumpe steigert. In der vorliegenden Arbeit wurden die Strukturwirkungsbeziehungen der Wechselwirkung zwischen TPGS und P-gp und der Mechanismus, der der Hemmung zu Grunde liegt, untersucht, um die TPGS Struktur zu optimieren. Die P-gp Hemmung durch TPGS konnte durch Variation der PEG Kettenlänge erhöht werden, wobei der optimale Hemmeffekt bei einem PEG Molekulargewicht von ungefähr 1500 Da beobachtet wurde. Des weiteren steigerten auch Modifikationen des hydrophoben Molekülteils, wie der Austausch des Alpha-Tocopherol-Restes durch Cholesterol, das Hemmpotenzial von TPGS. Die inhibitorische Wirkung der TPGS Derivate korreliert nicht mit ihren physikochemischen Eigenschaften wie Molekulargewicht, Molekülvolumen oder Lipophilie. In Übereinstimmung mit diesen Ergebnissen konnte eine unspezifische Änderung der Membranumgebung von P-gp in Elektronenspinresonanz-Untersuchungen ausgeschlossen werden. Für TPGS konnte gezeigt werden, dass es selbst kein Substrat von P-gp ist und nicht mit einer der transportaktiven Bindungsstellen von P-gp, im Sinne einer kompetitiven Hemmung interagiert. Vielmehr korreliert der inhibitorische Effekt mit der Hemmung der Substrat-induzierten ATPase Aktivität, was darauf hinweißt, dass diese Depletion der Energiequelle der Effluxpumpe ein essentieller Bestandteil des Hemmmechanismus von TPGS ist

Soluplus® as an effective absorption enhancer of poorly soluble drugs in vitro and in vivo

a b s t r a c t As many new active pharmaceutical ingredients are poorly water soluble, solubility enhancers are one possibility to overcome the hurdles of drug dissolution and absorption in oral drug delivery. In the present work a novel solubility enhancing excipient (Soluplus Ò ) was tested for its capability to improve intestinal drug absorption. BCS class II compounds danazol, fenofibrate and itraconazole were tested both in vivo in beagle dogs and in vitro in transport experiments across Caco-2 cell monolayers. Each drug was applied as pure crystalline substance, in a physical mixture with Soluplus Ò , and as solid solution of the drug in the excipient. In the animal studies a many fold increase in plasma AUC was observed for the solid solutions of drug in Soluplus Ò compared to the respective pure drug. An effect of Soluplus Ò in a physical mixture with the drug could be detected for fenofibrate. In vitro transport studies confirm the strong effect of Soluplus Ò on the absorption behavior of the three tested drugs. Furthermore, the increase of drug flux across Caco-2 monolayer is correlating to the increase in plasma AUC and C max in vivo. For these poorly soluble substances Soluplus Ò has a strong potential to improve oral bioavailability. The applicability of Caco-2 monolayers as tool for predicting the in vivo transport behavior of the model drugs in combination with a solubility enhancing excipient was shown. Also the improvement of a solid dispersion compared to physical mixtures of the drugs and the excipient was correctly reflected by Caco-2 experiments. In the case of fenofibrate the possible improvement by a physical mixture was demonstrated, underscoring the value of the used tool as alternative to animal studies

Modulation des apikalen Effluxtransporters P-glykoprotein durch Vitamin E TPGS:Struktur-Wirkungs-Beziehungen und Hemmmechanismus

The non-ionic surfactant D-alpha-tocopheryl poly(ethylene glycol 1000) succinate (TPGS 1000) has been previously shown to increase oral bioavailability of P-glycoprotein (P-gp) substrates by modulating activity of the efflux pump. In the present thesis structure activity relationship of the interaction and possible mechanism of inhibition of the efflux transporter were investigated to optimize the TPGS structure. P-gp inhibitory activity of TPGS could be increased by modifying the length of the PEG chain with optimal inhibition being achieved at a PEG molecular weight of ~1500 Da. The inhibitory effect of TPGS on P-gp could be also increased by modifications to the hydrophobic part of the molecule, such as the exchange of the alpha-tocopherol moiety for cholesterol. The inhibitory activity of the TPGS analogues does not correlate with their physicochemical properties such as molecular weight, molecule volume, or lipophilicity. In agreement with these findings, an unspecific alteration of the P-gp membrane environment could be ruled out in electron spin resonance experiments. TPGS was shown not to be a P-gp substrate itself nor does it interact with one of the transport active drug binding sites of P-gp to competitively block drug efflux. Rather, the inhibitory effect of the TPGS analogues correlates with their inhibition of substrate induced ATPase activity, indicating that the depletion of the energy source of the efflux pump is an integral part in the inhibitory mechanism of TPGS.Für das nicht ionische Tensid D-Alpha-tocopheryl poly(ethylene glycol 1000) succinat (TPGS 1000) wurde kürzlich gezeigt, dass es die orale Bioverfügbarkeit von Substraten des Effluxtransporters P-Glykoprotein (P-gp) durch Hemmung der Effluxpumpe steigert. In der vorliegenden Arbeit wurden die Strukturwirkungsbeziehungen der Wechselwirkung zwischen TPGS und P-gp und der Mechanismus, der der Hemmung zu Grunde liegt, untersucht, um die TPGS Struktur zu optimieren. Die P-gp Hemmung durch TPGS konnte durch Variation der PEG Kettenlänge erhöht werden, wobei der optimale Hemmeffekt bei einem PEG Molekulargewicht von ungefähr 1500 Da beobachtet wurde. Des weiteren steigerten auch Modifikationen des hydrophoben Molekülteils, wie der Austausch des Alpha-Tocopherol-Restes durch Cholesterol, das Hemmpotenzial von TPGS. Die inhibitorische Wirkung der TPGS Derivate korreliert nicht mit ihren physikochemischen Eigenschaften wie Molekulargewicht, Molekülvolumen oder Lipophilie. In Übereinstimmung mit diesen Ergebnissen konnte eine unspezifische Änderung der Membranumgebung von P-gp in Elektronenspinresonanz-Untersuchungen ausgeschlossen werden. Für TPGS konnte gezeigt werden, dass es selbst kein Substrat von P-gp ist und nicht mit einer der transportaktiven Bindungsstellen von P-gp, im Sinne einer kompetitiven Hemmung interagiert. Vielmehr korreliert der inhibitorische Effekt mit der Hemmung der Substrat-induzierten ATPase Aktivität, was darauf hinweißt, dass diese Depletion der Energiequelle der Effluxpumpe ein essentieller Bestandteil des Hemmmechanismus von TPGS ist

Dual flow bioreactor with ultrathin microporous TEER sensing membrane for evaluation of nanoparticle toxicity

Permeability studies across biological barriers are of primary importance in drug delivery as well as in toxicology when investigating the absorption and translocation of a substance. The study of nanomaterial interaction with epithelial barriers is of particular interest given their growing use in nanomedicine as well as concerns about their potential hazard. Here we describe the design and fabrication of a new bioreactor with an ultrathin microporous sensing support for the study of nanoparticle toxicity in intestinal epithelial cells in conditions which better recapitulate the physiological environment. Thanks to the integration of 4 electrodes in the microporous membrane, the system allows real-time and continuous sensing of TEER (trans epithelial electrical resistance) during flow without interruption or perturbation of experiments. The TEER bioreactor was tested using Caco-2 cells as an in vitro model of intestinal epithelia. When exposed to silver nanoparticles, which are known to be toxic, the embedded electrodes enabled non-invasive evaluation of barrier impairment over time. This device can be used to study barrier integrity and the kinetics of nanomaterial induced damage to epithelial barriers in physiologically relevant conditions

Crossing biological barriers for advanced drug delivery.

This special issue compiles invited and contributed papers of the 9th International Conference and Workshop "Biological Barriers", 29 February-9 March 2012 at Saarland University, Saarbrücken Germany

A 3D co-culture of three human cell lines to model the inflamed intestinal mucosa for safety testing of nanomaterials.

Oral exposure to nanomaterials is a current concern, asking for innovative biological test systems to assess their safety, especially also in conditions of inflammatory disorders. Aim of this study was to develop a 3D intestinal model, consisting of Caco-2 cells and two human immune cell lines, suitable to assess nanomaterial toxicity, in either healthy or diseased conditions. Human macrophages (THP-1) and human dendritic cells (MUTZ-3) were embedded in a collagen scaffold and seeded on the apical side of transwell inserts. Caco-2 cells were seeded on top of this layer, forming a 3D model of the intestinal mucosa. Toxicity of engineered nanoparticles (NM101 TiO2, NM300 Ag, Au) was evaluated in non-inflamed and inflamed co-cultures, and also compared to non-inflamed Caco-2 monocultures. Inflammation was elicited by IL-1β, and interactions with engineered NPs were addressed by different endpoints. The 3D co-culture showed well preserved ultrastructure and significant barrier properties. Ag NPs were found to be more toxic than TiO2 or Au NPs. But once inflamed with IL-1β, the co-cultures released higher amounts of IL-8 compared to Caco-2 monocultures. However, the cytotoxicity of Ag NPs was higher in Caco-2 monocultures than in 3D co-cultures. The naturally higher IL-8 production in the co-cultures was enhanced even further by the Ag NPs. This study shows that it is possible to mimic inflamed conditions in a 3D co-culture model of the intestinal mucosa. The fact that it is based on three easily available human cell lines makes this model valuable to study the safety of nanomaterials in the context of inflammation