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

Designing chitosan-tripolyphosphate microparticles with desired size for specific pharmaceutical or forensic applications

Chitosan (CS) is a natural cationic polymer obtained by the partial N-deacetylation of chitin. Chitosan microparticles can be prepared by cross-linking with tripolyphosphate (TPP) via the ionic interaction between positively charged amino groups (CS) and negatively charged counter ions (TPP). This can be controlled by the charge density of CS and TPP, which depend on the pH and ionic strength of the solution. The purpose of this study is to investigate the combined effects of three independent variables (pH, ionic strength and CS: TPP ratio) on three important physico-chemical properties (viscosity, zeta potential and particle size) during the preparation of microparticles. CS: TPP microparticles were prepared using experimental design and equations were generated and used to predict relative viscosity, zeta potential and particle size under different conditions. This gives us the ability to design tuneable CS-TPP microparticles with desired size for specific pharmaceutical or forensic applications e.g. latent fingerprint visualisation

Discovery of the inhibitory effect of a phosphatidylinositol derivative on P-glycoprotein by virtual screening followed by <i>in vitro</i> cellular studies

P-glycoprotein is capable of effluxing a broad range of cytosolic and membrane penetrating xenobiotic substrates, thus leading to multi-drug resistance and posing a threat for the therapeutic treatment of several diseases, including cancer and central nervous disorders. Herein, a virtual screening campaign followed by experimental validation in Caco-2, MDKCII, and MDKCII mdr1 transfected cell lines has been conducted for the identification of novel phospholipids with P-gp transportation inhibitory activity. Phosphatidylinositol-(1,2-dioctanoyl)-sodium salt (8∶0 PI) was found to significantly inhibit transmembrane P-gp transportation in vitro in a reproducible-, cell line-, and substrate-independent manner. Further tests are needed to determine whether this and other phosphatidylinositols could be co-administered with oral drugs to successfully increase their bioavailability. Moreover, as phosphatidylinositols and phosphoinositides are present in the human diet and are known to play an important role in signal transduction and cell motility, our finding could be of substantial interest for nutrition science as well

Profiles of Multidrug Resistance Protein-1 in the Peripheral Blood Mononuclear Cells of Patients with Refractory Epilepsy

BACKGROUND: About one third of patients with epilepsy become refractory to therapy despite receiving adequate medical treatment, possibly from multidrug resistance. P-glycoprotein, encoded by multidrug resistance protein-1 (MDR1) gene, at the blood brain barrier is considered as a major factor mediating drug efflux and contributing to resistance. Given that peripheral blood mononuclear cells (PBMNCs) express MDR1, we investigated a MDR1 status of PBMNCs in various subsets of epilepsy patients and demonstrated their association with clinical characteristics. METHODOLOGY/PRINCIPAL FINDINGS: Clinical and MDR1 data were collected from 140 patients with epilepsy, 30 healthy volunteers, and 20 control patients taking anti-epileptic drugs. PBMNCs were isolated, and basal MDR1 levels and MDR1 conformational change levels were measured by flow cytometry. MDR1 profiles were analyzed according to various clinical parameters, including seizure frequency and number of medications used in epilepsy patients. Epilepsy patients had a higher basal MDR1 level than non-epilepsy groups (p<0.01). Among epilepsy patients, there is a tendency for higher seizure frequency group to have higher basal MDR1 level (p = 0.059). The MDR1 conformational change level was significantly higher in the high-medication-use group than the low-use group (p = 0.028). Basal MDR1 (OR = 1.16 [95% CI: 1.060-1.268]) and conformational change level (OR = 1.11 [95% CI: 1.02-1.20]) were independent predictors for seizure frequency and number of medications, respectively. CONCLUSIONS/SIGNIFICANCE: The MDR1 profile of PBMNCs is associated with seizure frequency and medication conditions in patients with epilepsy

Natural and Synthetic Polymers as Inhibitors of Drug Efflux Pumps

Inhibition of efflux pumps is an emerging approach in cancer therapy and drug delivery. Since it has been discovered that polymeric pharmaceutical excipients such as Tweens® or Pluronics® can inhibit efflux pumps, various other polymers have been investigated regarding their potential efflux pump inhibitory activity. Among them are polysaccharides, polyethylene glycols and derivatives, amphiphilic block copolymers, dendrimers and thiolated polymers. In the current review article, natural and synthetic polymers that are capable of inhibiting efflux pumps as well as their application in cancer therapy and drug delivery are discussed

Improving the biopharmaceutical attributes of mangiferin using vitamin E-TPGS co-loaded self-assembled phosholipidic nano-mixed micellar systems

The current research work encompasses the development, characterization, and evaluation of self-assembled phospholipidic nano-mixed miceller system (SPNMS) of a poorly soluble BCS Class IV xanthone bioactive, mangiferin (Mgf) functionalized with co-delivery of vitamin E TPGS. Systematic optimization using I-optimal design yielded self-assembled phospholipidic nano-micelles with a particle size of  80% of drug release in 15 min. The cytotoxicity and cellular uptake studies performed using MCF-7 and MDA-MB-231 cell lines demonstrated greater kill and faster cellular uptake. The ex vivo intestinal permeability revealed higher lymphatic uptake, while in situ perfusion and in vivo pharmacokinetic studies indicated nearly 6.6- and 3.0-folds augmentation in permeability and bioavailability of Mgf. In a nutshell, vitamin E functionalized SPNMS of Mgf improved the biopharmaceutical performance of Mgf in rats for enhanced anticancer potency

Effects of polyethylene glycols on intestinal efflux pump expression and activity in Caco-2 cells

The present study was planned to investigate the influence of polyethylene glycols (PEGs) on the activity and expression of P-glycoprotein (P-gp). Sub-toxic concentrations of PEGs in Caco-2 cells were determined using the MTT test assay. Then the measurement of Rhodamine-123 (Rho-123) uptake, a P-gp fluorescence substrate, in Caco-2 cells confronting PEG 400 (1% and 2% w/v), PEG 4000 (2% and 4% w/v), PEG 6000 (2% and 4% w/v), PEG 10000 (2% and 4% w/v), PEG 15000 (1% and 2% w/v), and PEG 35000 (2% and 4% w/v) overnight was taken to elucidate whether non-toxic concentrations of PEGs are able to impact P-gp activity. Furthermore, western blotting was carried out to investigate P-gp protein expression. The results showed that PEG 400 at concentrations of 1% (w/v) and 2% (w/v) and PEG 6000 at the concentration of 4% (w/v) are notably capable of blocking P-gp. Based on the obtained results it is concluded that the mentioned excipients could be used to obstruct P-gp efflux transporter in order to increase the bioavailability of co-administered substrate drug