Prolongation of the gastric residence time of caffeine after administration in fed state: Comparison of effervescent granules with an extended release tablet.

The aim of the present study was to investigate the gastroretentive capacity of different formulation principles. This was indirectly determined by the absorption behavior of caffeine from the dosage forms. A slow and continuous appearance of caffeine in the saliva of healthy volunteers was used as a parameter for a prolonged gastric retention time. For this purpose, a four-way study was conducted with twelve healthy volunteers using the following test procedures: (1) Effervescent granules with 240 mL of still water administered in fed state, (2) effervescent granules with 20 mL of still water in fed state, (3) extended release (ER) tablet with 240 mL of still water in fed state, and (4) effervescent granules with 240 mL of still water in fasted state. The initial rise of the caffeine concentrations was more pronounced after the intake of the effervescent granules in the fed state compared to that of the ER tablets. However, tmax tended to be shorter in the fed study arms following administration of the ER tablet compared to the granules. Overall, the application of active pharmaceutical ingredients formulated as effervescent granules seems to be a promising approach to increase their gastric residence time after intake in fed state.

Comparing the gastric emptying of 240 mL and 20 mL water by MRI and caffeine salivary tracer technique.

Gastrointestinal fluid volumes are a crucial parameter for dissolution and absorption of orally taken medications. Most often 240 mL are used in clinical standard setups. Nonetheless, surveys in patient populations revealed dramatically lower volumes for intake of oral medications in real life and even in some clinical studies reduced fluid volumes are common. These reductions might have serious impact on pharmacokinetics. Thus, it was the aim of this study to compare the gastric emptying of 240 mL and 20 mL of water in 8 healthy volunteers. For investigation of gastric fluid volumes Magnetic Resonance Imaging with strongly T2 weighted sequences was used. Gastric emptying was additionally quantified via caffeine pharmacokinetics measured in saliva. The absolute gastric volumes after intake of 240 mL or 20 mL obviously differed by factor 10 but relative gastric emptying expressed as fraction per time was nearly comparable. Only slighter slower emptying after intake of 20 mL was observed. Salivary caffeine pharmacokinetics representing mass transfer from stomach to small intestine after intake of different volumes did not differ. The absorbed caffeine fraction and emptied gastric volume fraction correlated well after intake of 240 mL, but not after intake of 20 mL, indicating a higher influence of secretion on gastric volume measurements after intake of smaller volumes. Relative gastric emptying as measured with MRI and salivary caffeine method was only slightly delayed, thus transfer of orally administered drug fraction could be comparable even with lower fluid intake as can be seen by comparable caffeine pharmacokinetics. Nonetheless, the considerably reduced volumes might interfere with dissolution and absorption.

Individualized, Additively Manufactured Drug-Releasing External Ear Canal Implant for Prevention of Postoperative Restenosis: Development, In Vitro Testing, and Proof of Concept in an Individual Curative Trial.

Postoperative restenosis in patients with external ear canal (EEC) atresia or stenosis is a common complication following canaloplasty. Our aim in this study was to explore the feasibility of using a three dimensionally (3D)-printed, patient-individualized, drug ((dexamethasone (DEX)), and ciprofloxacin (cipro))-releasing external ear canal implant (EECI) as a postoperative stent after canaloplasty. We designed and pre-clinically tested this novel implant for drug release (by high-performance liquid chromatography), biocompatibility (by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay), bio-efficacy (by the TNF-α (tumor necrosis factor-alpha)-reduction test (DEX) and inhibition zone test (for cipro)), and microbial contamination (formation of turbidity or sediments in culture medium). The EECI was implanted for the first time to one patient with a history of congenital EEC atresia and state after three canaloplasties due to EEC restenosis. The preclinical tests revealed no cytotoxic effect of the used materials; an antibacterial effect was verified against the bacteria Staphylococcus aureus and Pseudomonas aeruginosa, and the tested UV-irradiated EECI showed no microbiological contamination. Based on the test results, the combination of silicone with 1% DEX and 0.3% cipro was chosen to treat the patient. The EECI was implantable into the EEC; the postoperative follow-up visits revealed no otogenic symptoms or infections and the EECI was explanted three months postoperatively. Even at 12 months postoperatively, the EEC showed good epithelialization and patency. Here, we report the first ever clinical application of an individualized, drug-releasing, mechanically flexible implant and suggest that our novel EECI represents a safe and effective method for postoperatively stenting the reconstructed EEC.

The challenges of drug delivery to the esophagus and how to overcome them.

INTRODUCTION: The incidence of esophageal diseases such as eosinophilic esophagitis, gastroesophageal reflux disease, or esophageal carcinoma has increased significantly in recent years. There is a lack of suitable therapeutic options to enable effective therapy. AREAS COVERED: This review addresses the challenges of drug targeting to the esophagus. It describes the physiology of the esophagus, physiological parameters relevant for drug targeting and the pathophysiology of selected diseases of the esophagus. Furthermore, conventional dosage forms such as glucocorticoid-containing solutions or suspensions, inhaler devices and orodispersible tablets are discussed. Innovative drug delivery systems such as stents, mucoadhesive films and 3D printed devices are also presented. EXPERT OPINION: Drug delivery to the esophagus presents challenges, especially in terms of the short transit time and the rapid clearance. While conventional dosage forms such as solutions or suspensions offer the advantage of being available quickly and patient-specific, innovative drug delivery systems address the problem of the short transit time and can thus enable long-lasting exposure.

Impact of gastrointestinal tract variability on oral drug absorption and pharmacokinetics: An UNGAP review.

The absorption of oral drugs is frequently plagued by significant variability with potentially serious therapeutic consequences. The source of variability can be traced back to interindividual variability in physiology, differences in special populations (age- and disease-dependent), drug and formulation properties, or food-drug interactions. Clinical evidence for the impact of some of these factors on drug pharmacokinetic variability is mounting: e.g. gastric pH and emptying time, small intestinal fluid properties, differences in pediatrics and the elderly, and surgical changes in gastrointestinal anatomy. However, the link of colonic factors variability (transit time, fluid composition, microbiome), sex differences (male vs. female) and gut-related diseases (chronic constipation, anorexia and cachexia) to drug absorption variability has not been firmly established yet. At the same time, a way to decrease oral drug pharmacokinetic variability is provided by the pharmaceutical industry: clinical evidence suggests that formulation approaches employed during drug development can decrease the variability in oral exposure. This review outlines the main drivers of oral drug exposure variability and potential approaches to overcome them, while highlighting existing knowledge gaps and guiding future studies in this area.

In Vitro and In Vivo Evaluation of 3D Printed Capsules with Pressure Triggered Release Mechanism for Oral Peptide Delivery.

In this study a 3D printed capsule designed to break from the physiological pressures in the antropyloric region was evaluated for its ability to deliver the synthetic octapeptide octreotide in beagle dogs when co-formulated with the permeation enhancer sodium caprate. The pressure sensitive capsules were compared to traditional enteric coated hard gelatin capsules and enteric coated tablets. Paracetamol, which is completely absorbed in dogs, was included in the formulations and used as an absorption marker to give information about the in vivo performance of the dosage forms. The pressure sensitive capsules released drug in 50% of the dogs. In the cases where drug was released, there was no difference in octreotide bioavailability or Cmax compared to the enteric coated dosage forms. When comparing all dosage forms, a correlation was seen between paracetamol Cmax and octreotide bioavailability, suggesting that a high drug release rate may be beneficial for peptide absorption when delivered together with sodium caprate.

Effect of Coadministered Water on the In Vivo Performance of Oral Formulations Containing N-Acetylcysteine: An In Vitro Approach Using the Dynamic Open Flow-Through Test Apparatus.

The drug plasma profile after oral administration of immediate release dosage forms can be affected by the human gastrointestinal physiology, the formulation, and the drug itself. In this work, we investigated the in vivo and in vitro performance of two formulations (granules vs. tablet) containing the highly soluble drug N-Acetylcysteine (BCS class I). Thereby, special attention was paid to the effect of the dosage form and the coadministration of water on drug release. Interestingly, the in vivo results from a pharmacokinetic study with 11 healthy volunteers indicated that the drug plasma concentrations were comparable for the tablet given with water as well as for the granules given with and without water. In order to mechanistically understand this outcome, we used a biorelevant dissolution test device, the dynamic open flow-through test apparatus. With the aid of this test apparatus, we were able to simulate biorelevant parameters, such as gastric emptying, hydrodynamic flow as well as physical stress. By this, it was possible to mimic the intake conditions of the clinical trial (i.e., drug intake with and without water). Whereas the experiments in the USP paddle apparatus revealed differences between the two formulations, we could not observe significant differences in the release profiles of the two formulations by using the dynamic open flow-through test apparatus. Even by considering the different intake conditions, drug release was slow and amounted to around 30% until simulated gastric emptying. These results suggest that dissolution was irrespective of coadministered water and the formulation. Despite the high aqueous solubility of N-Acetylcysteine, the limiting factor for drug release was the slow dissolution rate in relation to the gastric emptying rate under simulated gastric conditions. Thus, in case of administration together with water, large amounts of the drug are still present in the stomach even after complete gastric emptying of the water. Consequently, the absorption of the drug is largely controlled by the nature of gastric emptying of the remaining drug. The data of this study indicated that the water emptying kinetics are only determining drug absorption if drug release is rapid enough. If this is not the case, physiological mechanisms, such as the migrating motor complex, play an important role for oral drug delivery.

Muscle Injury After Intramuscular Administration of Diclofenac: A Case Report Supported by Magnetic Resonance Imaging.

Intramuscular injection of diclofenac is still frequently practiced, although there is ample evidence that the risk of local tissue intolerability is highly underestimated. The aim of this study was to evaluate local toxicity in a patient using magnetic resonance imaging. A patient who gave written informed consent received a medically indicated intramuscular administration of diclofenac 75 mg/2 mL. Simultaneously with magnetic resonance imaging of the depot, a clinical-chemical evaluation and quantification of diclofenac in plasma was performed. A manifold enhancement of the T2-weighted magnetic resonance signal was observed in a muscle area of approximately 60 mL volume, with maximum signal intensity 30 min after injection, the time of maximum diclofenac plasma exposure. Plasma creatine kinase activity was elevated approximately sixfold within 8 h and normalized within 1 week, whereas the magnetic resonance enhancement disappeared within 5 weeks. Interestingly, the patient did not complain about any clinical symptoms at the injection site. Asymptomatic tissue injury after intramuscular injection of diclofenac, caused by intramuscular dosing, can be reliably evaluated by magnetic resonance imaging and should be applied early during the development of parenteral dosage forms. Clinical Trials Registration Number: BB130/16 (Ethics Committee of the University Medicine Greifswald).

Tumour-specific delivery of siRNA-coupled superparamagnetic iron oxide nanoparticles, targeted against PLK1, stops progression of pancreatic cancer.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies and is projected to be the second leading cause of cancer-related death by 2030. Despite extensive knowledge and insights into biological properties and genetic aberrations of PDAC, therapeutic options remain temporary and ineffective. One plausible explanation for the futile response to therapy is an insufficient and non-specific delivery of anticancer drugs to the tumour site. DESIGN: Superparamagnetic iron oxide nanoparticles (SPIONs) coupled with siRNA directed against the cell cycle-specific serine-threonine-kinase, Polo-like kinase-1 (siPLK1-StAv-SPIONs), could serve a dual purpose for delivery of siPLK1 to the tumour and for non-invasive assessment of efficiency of delivery in vivo by imaging the tumour response. siPLK1-StAv-SPIONs were designed and synthesised as theranostics to function via a membrane translocation peptide with added advantage of driving endosomal escape for mediating transportation to the cytoplasm (myristoylated polyarginine peptides) as well as a tumour-selective peptide (EPPT1) to increase intracellular delivery and tumour specificity, respectively. RESULTS: A syngeneic orthotopic as well as an endogenous cancer model was treated biweekly with siPLK1-StAv-SPIONs and tumour growth was monitored by small animal MRI. In vitro and in vivo experiments using a syngeneic orthotopic PDAC model as well as the endogenous LSL-KrasG12D, LSL-Trp53R172H, Pdx-1-Cre model revealed significant accumulation of siPLK1-StAv-SPIONs in PDAC, resulting in efficient PLK1 silencing. Tumour-specific silencing of PLK1 halted tumour growth, marked by a decrease in tumour cell proliferation and an increase in apoptosis. CONCLUSIONS: Our data suggest siPLK1-StAv-SPIONs with dual specificity residues for tumour targeting and membrane translocation to represent an exciting opportunity for targeted therapy in patients with PDAC.

Exosomal particles secreted by prostate cancer cells are potent mRNA and protein vehicles for the interference of tumor and tumor environment.

BACKGROUND: Remodeling of the tumor environment and the modulation of tumor associated non-malignant cells are essential events in tumor progression. Exosomes are small membranous vesicles of 50-150 nm in diameter, which are secreted into the extracellular space and supposedly serve as vehicles for signal and effector molecules to modulate adjacent target cells. We characterized the mRNA and protein composition as well as cellular functions of prostate cancer cell-derived exosomes. METHODS: Exosomes were prepared from prostate cancer cell culture supernatant by ultracentrifugation and subsequently characterized by dynamic light scattering and electron microscopy. Exosomal mRNA and protein composition were analyzed by DNA microarrays and gel electrophoresis coupled with mass spectrometry. Physiological effects of exosomes were studied by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase release cell assays. Using a SILAC approach, putative uptake of exosomal human proteins in canine cells and canine de novo synthesis of proteins specified by exosome-transferred human mRNA was analyzed in MDCK cells via mass spectrometry. RESULTS: Preparations of exosomes revealed typical cup shaped particles of 150 nm in diameter. Analysis of mRNA and protein composition of exosomes exhibited a wide range of mRNA and protein species. Interestingly, the packaging of at least small proteins into exosomes was apparently unspecific, as shown with the example of two model proteins. In cell culture incubation experiments exosomal preparations of prostate cancer cells caused anti-proliferative effects. MS analysis revealed the uptake of exosomal human proteins into canine cells after 6 hr of incubation. CONCLUSIONS: The results reveal a distinct exosomal functionality in the modulation of the prostatic tumor adjacent environment. The multitude of translocated factors implies the induction of numerous effects in tumor-associated target cells, including impact on cellular growth.

In vitro evaluation of paclitaxel coatings for delivery via drug-coated balloons.

Lately, drug-coated balloons have been introduced in interventional cardiology as an approach to treat occluded blood vessel. They were developed for the rapid transfer of antiproliferative drugs during the angioplasty procedure in stenosed vessels with the intent to reduce the risk of restenosis. In this study five different paclitaxel (PTX) balloon coatings were tested in vitro in order to examine how solvents and additives influence coating stability and drug transfer rates. PTX-coated balloons were advanced through a guiding catheter and a simulated coronary artery pathway under perfusion and were then inflated in a hydrogel acceptor compartment. The fractions transferred to the gel, remaining on the balloon and the PTX lost in the simulated coronary pathway were then analysed. The results obtained suggest that the solvent used for the coating process strongly influences the surface structure and the stability of the coating. Ethanol/water and acetone based PTX coatings showed the lowest drug transfer rates to the simulated vessel wall (both <1%) due to their high drug losses during the prior passage through the coronary artery model (more than 95%). Balloons coated with PTX from ethyl acetate-solutions showed smaller drug loss (83%±9%), but most of the remaining PTX was not transferred (mean balloon residue approximately 15%). Beside the solvent, the use of additives seemed to have a great impact on transfer properties. The balloon pre-treatment with a crosslinked polyvinylpyrrolidone (PVP) film was able to increase the PTX transfer rate from less than 1% (without PVP) to approximately 6%. The best results in this study were obtained for balloon coatings with commercially available SeQuent© Please balloons containing the contrast agent iopromide. For this formulation drug transfer rates of approximately 17% were determined. Fluorescence microscopic imaging could visualize the particulate transfer of labelled PTX from the balloon surface during dilatation. The findings of this study underline the importance of drug adhesion and coating stability for the efficiency of PTX transfer.

Characterization of the intestinal and hepatic uptake/efflux transport of the magnetic resonance imaging contrast agent gadolinium-ethoxylbenzyl-diethylenetriamine-pentaacetic acid.

OBJECTIVES: The objectives of the study were to measure the pharmacokinetics and liver enhancement of gadoxetate (gadolinium-ethoxylbenzyl-diethylenetriamine-pentaacetic acid [Gd-EOB-DTPA], Eovist, Primovist) after oral and intravenous administration in wild-type and (multidrug resistance-associated protein 2) Mrp2-deficient rats and to evaluate the in vitro transport of the contrast agent via intestinal and hepatic transporter proteins. MATERIALS AND METHODS: Gadolinium-ethoxylbenzyl-diethylenetriamine-pentaacetic acid-enhanced magnetic resonance imaging and pharmacokinetics of Gd-EOB-DTPA after intravenous and oral administration were evaluated in wild-type and Mrp2-deficient rats using T1-weighted magnetic resonance imaging and a validated liquid chromatography-mass spectrometry method, respectively. Cellular uptake of Gd-EOB-DTPA was measured in stably transfected human embrionic kidney 293-cells expressing oragnic anion-transporting polypeptide 1A2 or organic cation transporter 3 and Madin Darby canine kidney 2-cells expressing apical sodium dependent bile acid transporter. The affinity to MRP2 and multidrug resistance-associated protein 3 was measured using inside-out vesicles. RESULTS: In vitro, Gd-EOB-DTPA was demonstrated to be a substrate for OATP1A2 (mean [SD] of the Michaelis-Menten constant [K(m)], 1.0 [0.4] mmol/L; mean [SD] of the maximal uptake rate [V(max)], 101.3 [21.1] pmol/mg per minute), MRP2 (K(m), 1.0 [0.5] mmol/L; V(max), 86.8 [31.1] pmol/mg per minute), and multidrug resistance-associated protein 3 (K(m), 1.8 [0.3] mmol/L; V(max), 116 [15.9] pmol/mg per minute) but not for the apical sodium-dependent bile acid transporter and organic cation transporter 3. After the oral administration to the wild-type animals, Gd-EOB-DTPA was considerably absorbed from the small intestine (bioavailability, approximately 17%) and predominately eliminated via feces after intravenous dosing (approximately 96%). In the Mrp2-deficient rats, oral bioavailability increased to approximately 21% and Gd-EOB-DTPA was exclusively excreted into urine. Magnetic resonance enhancement of the liver was significantly prolonged in the Mrp2-deficient rats compared with the wild-type rats (mean [SD] area under the curve0-90, 36.4 [8.5] vs 14.8 [10.3] arbitary units per minute; P = 0.003; time to maximum plasma concentration, 48.6 [23.8] vs 6.0 [3.1] minutes; P = 0.001). CONCLUSIONS: The nonmetabolized Gd-EOB-DTPA may have some potentials to be used as a probe-contrast agent to evaluate transporter-mediated mechanisms along the enterohepatic absorption route for drugs by functional visualization in vivo.

In vitro dissolution testing of drug-eluting stents.

Drug-eluting stents (DES) have revolutionized the treatment of coronary artery blockage by tremendously reducing the rate of in-stent restenosis and the necessity of repeat revascularization compared to bare-metal stents. They are also gaining increasing importance in other medical fields such as the treatment of certain localized tumors and in glaucoma therapy. DES generally contain most potent drugs, e.g. immunosuppressants or cytostatics, which are supposed to be released in a well controlled manner over time spans which are chosen according to disease progression. Typically, this means that fairly small amounts of drug are released over long periods of time. Therefore, quantification of in vivo plasma levels is often not feasible. Due to this limitation and the fact that tissue levels cannot be determined in humans, in vitro dissolution testing is one of the most powerful tools to gain insight into the release behaviour of DES. This article focuses on the methods for in vitro dissolution testing of DES which are available up to date and highlights the specific characteristics of drug release from stents arising from the composition and the in vivo localization of the dosage form.

Changes in recovery due to drug product matrix ageing as a source of mass imbalances.

An important quality feature of stability testing of drug products is mass balance. Besides several known or anticipated causes for mass imbalances, a further potential cause that has not yet been systematically assessed might be incomplete recovery due to the influence of matrix ageing. The genotoxic degradation product 4-chloroaniline (PCA) and the unstable drug substance estradiol (E2) that is known to be difficult to extract from matrices in low-dose solid formulations were chosen as examples. A marketed product containing E2 as well as two marketed products that potentially contain PCA were investigated together with experimental formulations containing E2 or PCA that were produced for this study. To accelerate drug product matrix ageing, samples were stored at different conditions for defined storage periods. PCA and E2 recovery was determined at all sampling time points, respectively. In comparison to unstressed samples, significant changes in recovery were observed in 67% of the formulations investigated. Consequently, the outlined procedure can be regarded as a promising approach to reveal potential reasons for mass imbalance.

Visualization of hepatic uptake transporter function in healthy subjects by using gadoxetic acid-enhanced MR imaging.

PURPOSE: To determine if genetic polymorphisms of liver-specific human organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 influence cellular uptake of gadoxetic acid in vitro and if functionally relevant polymorphisms are confounders for liver enhancement by gadoxetic acid in healthy subjects. MATERIALS AND METHODS: This study received ethics approval, and all subjects provided written informed consent. Cellular uptake of gadoxetic acid by OATP1B1 and OATP1B3 and their frequent genetic variants was measured by using stable transfected embryonic kidney HEK293 cells. Liver signal intensity at gadoxetic acid-enhanced MR imaging and pharmacokinetics of gadoxetic acid were evaluated in 36 healthy carriers of SLCO1B1/1B3 wild-type alleles (n = 10), SLCO1B1*1b/*1b (n = 8), SLCO1B1*15/*15 (n = 7), SLCO1B1*5/*15 (n = 1), SLCO1B1*1a/*5 (n = 6), and SLCO1B3*4/*4 (n = 4) by using T1-weighted MR imaging and liquid chromatography tandem mass spectrometry. RESULTS: Transport activity for gadoxetic acid was increased in cells transfected with SLCO1B1c.388A>G (12.8 pmol/[mg·min]6 3.53, P = .001) but decreased in cells with SLCO1B1c.388A>G/521T>C (3.11 pmol/[mg·min] ± 0.918, P = .004) compared with cells with nonvariant transporter (6.32 pmol/[mg·min] ± 2.73). Compared with activity of cells transfected with the nonvariant SLCO1B3 (7.43 pmol/[mg·min] ± 2.43), SLCO1B3c.699G>A was a gain-of-function variant (15.1 pmol/[mg·min] ± 5.52, P = .002), whereas SLCO1B3c.334T>G (0.364 pmol/[mg·min] ± 0.125, P = .0001) and SLCO1B3c.1564G>T (0.295 pmol/[mg·min] ± 0.247, P = .0001) were variants with lower function. Liver enhancement with gadoxetic acid was reduced in subjects with OATP1B1*1a/*5 compared with wild-type subjects and those with OATP1B1*1b/*1b (area under enhancement curve, 3-480 minutes in arbitrary units [au]; 20.7 au ± 6.85 vs 36.5 au ± 8.08 [P = .006] vs 34.6 au ± 8.92 [P = .026]). The OATP1B3*4 polymorphism was not of functional relevance. No pharmacokinetic characteristics of gadoxetic acid were influenced by genetic polymorphisms of OATP1B1 and OATP1B3. CONCLUSION: Liver-specific OATP1B1 and OATP1B3 are uptake carriers for gadoxetic acid in subjects. Genetic polymorphisms of OATP1B1 are signal confounders in gadoxetic acid-enhanced liver MR imaging.

A novel liquefied gas based oral controlled release drug delivery system for liquid drug formulations.

A novel liquefied gas based drug delivery system for the oral delivery of liquid and semi-solid drug formulations is presented. The capsule-shaped system is equipped with a capillary as an element controlling the release rate. The delivery mechanism is based on a constant vapor pressure produced by isopentane as a low-boiling liquefied gas. The liquid drug valproic acid (VA) was used as a model compound. The viscosity was increased by the addition of povidone (PVP). The VA-PVP gel exhibited pseudoplastic rheological properties, the shear rate was above 0.1s(-1), similar to a Newtonian liquid. The gels tested in the gas based delivery system provided near-zero-order release kinetics. The longest delivery time was up to ca. 8h. The system is characterized by high flexibility of the delivery rate, which can be achieved by adjusting system parameters such as the diameter and length of the capillary, the vapor pressure of the propellant and the viscosity of the drug formulation.

N-glycosylation of ABC transporters is associated with functional activity in sandwich-cultured rat hepatocytes.

Hepatobiliary elimination via canalicular efflux transport proteins plays a key role in the clearance of endo- and xenobiotics. Correct membrane localization and coordinated action of the transport systems are essential for vectorial transport of drugs from blood into the bile. While basolaterally localized uptake transporters are responsible for the inward transport of substances from the blood into the hepatocyte, apically expressed ATP-dependent transport proteins such as P-glycoprotein (P-gp), multidrug resistance-associated protein (Mrp2) and breast cancer resistance protein (Bcrp) mediate the outward efflux into the bile canaliculus. Using sandwich-cultured rat hepatocytes we have characterized the expression and maturation of P-gp, Mrp2 and Bcrp transport proteins as well as their transport function over several days. The re-differentiation of the hepatocytes, which only occurs in sandwich configuration involves de novo synthesis and subsequent posttranslational N-glycosylation of all three transport proteins. Only fully N-glycosylated isoforms of the transporters were associated with functional activity as visualized by excretion of specific fluorescent substrates into the canalicular network. However, in what way N-glycosylation affects the functional activity of the ABC transporters investigated remains to be determined.

Hepatic uptake of the magnetic resonance imaging contrast agent Gd-EOB-DTPA: role of human organic anion transporters.

Contrast-enhancing magnetic resonance imaging with the liver-specific agent gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) has been shown to improve the detection rate of focal lesions. There is evidence from preclinical studies that multidrug organic anion transporters are involved in hepatic uptake of Gd-EOB-DTPA. Therefore, we evaluated affinity of the contrast agent to human organic anion-transporting polypeptides (OATP1B1, OATP1B3, OATP2B1) and to the Na(+)/taurocholate cotransporting polypeptide (NTCP) using stable transfected human embryonic kidney (HEK) 293 cells. In competition assays, Gd-EOB-DTPA inhibited the uptake of bromosulfophthalein (BSP) by OATP1B1 (IC(50) = 0.6 mM) and OATP1B3 (IC(50) = 0.4 mM). In comparison, the IC(50) values for rifampicin were 11.9 (OATP1B1), 1.4 (OATP1B3), and 80.5 muM (OATP2B1), respectively. Uptake of BSP by OATP2B1, uptake of taurocholic acid by NTCP, and viability of all HEK cells were not influenced by Gd-EOB-DTPA in concentrations up to 10 mM. In uptake assays using a new liquid chromatography-tandem mass spectrometry method for quantification, Gd-EOB-DTPA was a substrate for OATP1B1 (K(m) = 0.7 mM, V(max) = 10.5 pmol/mg x min), OATP1B3 (K(m) = 4.1 mM, V(max) = 22.7 pmol/mg x min), and NTCP (K(m) = 0.04 mM, V(max) = 1.4 pmol/mg x min). The uptake by OATP2B1 was not different from the vector control. In conclusion, Gd-EOB-DTPA is a substrate of the liver-specific OATP1B1, OATP1B3, and NTCP.

Development of a vessel-simulating flow-through cell method for the in vitro evaluation of release and distribution from drug-eluting stents.

A novel in vitro dissolution test for drug-eluting stents (DES) based on the compendial flow-through cell was developed. The model contains an additional compartment simulating the vessel wall enabling the examination of drug release and distribution. The compartment consists of alginate hydrogel containing a central aperture forming the lumen for stent placement and media flow. The method was tested utilizing stents coated with hydrophilic (fluorescein sodium) and hydrophobic (triamterene) fluorescent model substances as well as the cytostatic drug doxorubicin hydrochloride and compared to standard dissolution methods. The results show the suitability of the developed method to observe drug release and distribution. The determination of model substance content in the compartments media, hydrogel and stent yielded differing distribution patterns for the model compounds and enabled the observation of redistribution processes. The dissolution profiles differed from the results of compendial dissolution testing. Besides lower endpoints and slower rises of media concentrations due to distribution into the hydrogel, the release rates from the stent coatings were altered. These findings emphasize the necessity to adapt dissolution testing for DES to the unique conditions influencing delivery to the vessel wall to learn more about local distribution and to anticipate the in vivo performance of DES.