Expression of MATE1, P-gp, OCTN1 and OCTN2, in epithelial and immune cells in the lung of COPD and healthy individuals.

BACKGROUND: Several inhaled drugs are dependent on organic cation transporters to cross cell membranes. To further evaluate their potential to impact on inhaled drug disposition, the localization of MATE1, P-gp, OCTN1 and OCTN2 were investigated in human lung. METHODS: Transporter proteins were analysed by immunohistochemistry in lung tissue from healthy subjects and COPD patients. Transporter mRNA was analysed by qPCR in lung tissue and in bronchoalveolar lavage (BAL) cells from smokers and non-smokers. RESULTS: We demonstrate for the first time MATE1 protein expression in the lung with localization to the apical side of bronchial and bronchiolar epithelial cells. Interestingly, MATE1 was strongly expressed in alveolar macrophages as demonstrated both in lung tissue and in BAL cells, and in inflammatory cells including CD3 positive T cells. P-gp, OCTN1 and OCTN2 were also expressed in the alveolar epithelial cells and in inflammatory cells including alveolar macrophages. In BAL cells from smokers, MATE1 and P-gp mRNA expression was significantly lower compared to cells from non-smokers whereas no difference was observed between COPD patients and healthy subjects. THP-1 cells were evaluated as a model for alveolar macrophages but did not reflect the transporter expression observed in BAL cells. CONCLUSIONS: We conclude that MATE1, P-gp, OCTN1 and OCTN2 are expressed in pulmonary lung epithelium, in alveolar macrophages and in other inflammatory cells. This is important to consider in the development of drugs treating pulmonary disease as the transporters may impact drug disposition in the lung and consequently affect pharmacological efficacy and toxicity.

Molecular data reveal a tropical freshwater origin of Naidinae (Annelida, Clitellata, Naididae).

The phylogenetic relationships within Naidinae (Annelida, Clitellata, Naididae) were investigated, using six molecular markers, both mitochondrial (12S rDNA, 16S rDNA, the COI gene) and nuclear (18S rDNA, 28S rDNA, the ITS region). Thirty-seven nominal species, representing 16 of the 22 genera recognized in the subfamily, were included, and the Nais communis/variabilis species complex was represented by six different morphotypes. Ten other species of Naididae were selected as outgroups. The data were analysed by Bayesian inference and Maximum Likelihood. The phylogeny corroborates monophyly of the Naidinae, and the separate status of the genus Pristina (Pristininae) and the Opistocystinae. Relationships within Naidinae are largely well supported, but in some parts unexpected: (1) A clade containing the largely tropical genera Dero and Branchiodrilus is sister to the rest of the subfamily, and together with a third tropical genus, Allonais, they form a basal paraphyly. All these genera show morphological adaptations to environmental hypoxia, leading to the conclusion that Naidinae originated in tropical freshwaters. (2) The genera Dero, Nais and Piguetiella are paraphyletic. (3) At least Branchiodrilus, Paranais, Chaetogaster, Nais, Stylaria appear to contain cryptic species. Morphological characters, especially those associated with chaetae, are to a great extent homoplastic within Naidinae, which certainly has contributed to the overall taxonomic confusion of this subfamily.

Best practice in placement of percutaneous endoscopic gastrostomy with jejunal extension tube for continuous infusion of levodopa carbidopa intestinal gel in the treatment of selected patients with Parkinson's disease in the Nordic region.

OBJECTIVE: Continuous infusion of levodopa carbidopa intestinal gel (LCIG) is associated with a significant improvement in the symptoms and quality of life of selected patients with advanced Parkinson's disease. Percutaneous endoscopic gastrostomy with jejunal extension (PEG/J) was first described in 1998 and has become the most common and standard technique for fixing the tubing in place for LCIG infusion. MATERIAL AND METHODS: A workshop was held in Stockholm, Sweden, to discuss the PEG/J placement for the delivery of LCIG in Parkinson's disease patients with the primary goal of providing guidance on best practice for the Nordic countries. RESULTS: Suggested procedures for preparation of patients for PEG/J placement, aftercare, troubleshooting and redo-procedures for use in the Nordic region are described and discussed. CONCLUSIONS: LCIG treatment administered through PEG/J-tubes gives a significant increase in quality of life for selected patients with advanced Parkinson's disease. Although minor complications are common, serious complications are infrequent, and the tube insertion procedures have a good safety record. Further development of delivery systems and evaluation of approaches designed to reduce the demand for redo endoscopy are required.

Cationic uremic toxins affect human renal proximal tubule cell functioning through interaction with the organic cation transporter.

Several organic cations, such as guanidino compounds and polyamines, have been found to accumulate in plasma of patients with kidney failure due to inadequate renal clearance. Here, we studied the interaction of cationic uremic toxins with renal organic cation transport in a conditionally immortalized human proximal tubule epithelial cell line (ciPTEC). Transporter activity was measured and validated in cell suspensions by studying uptake of the fluorescent substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium-iodide (ASP(+)). Subsequently, the inhibitory potencies of the cationic uremic toxins, cadaverine, putrescine, spermine and spermidine (polyamines), acrolein (polyamine breakdown product), guanidine, and methylguanidine (guanidino compounds) were determined. Concentration-dependent inhibition of ASP(+) uptake by TPA, cimetidine, quinidine, and metformin confirmed functional endogenous organic cation transporter 2 (OCT2) expression in ciPTEC. All uremic toxins tested inhibited ASP(+) uptake, of which acrolein required the lowest concentration to provoke a half-maximal inhibition (IC50 = 44 ± 2 µM). A Dixon plot was constructed for acrolein using three independent inhibition curves with 10, 20, or 30 µM ASP(+), which demonstrated competitive or mixed type of interaction (K i = 93 ± 16 µM). Exposing the cells to a mixture of cationic uremic toxins resulted in a more potent and biphasic inhibitory response curve, indicating complex interactions between the toxins and ASP(+) uptake. In conclusion, ciPTEC proves a suitable model to study cationic xenobiotic interactions. Inhibition of cellular uptake transport was demonstrated for several uremic toxins, which might indicate a possible role in kidney disease progression during uremia.

New insights into the carrier-mediated transport of estrone-3-sulfate in the Caco-2 cell model.

The current studies were undertaken to gain new insights into the interplay and mechanism of membrane transporters involved in the permeability of estrone-3-sulfate (E1S) in Caco-2 cells cultured either on the bottom of multiwell plastic dishes or on filter support. We demonstrate that Caco-2 cells from the "Deutsche sammlung von mikroorganismen und zellkulturen" (DSMZ) exhibit extensive and consistent carrier-mediated uptake of [(3)H]-E1S after a culture period of 11-13 days. The kinetic characterization, the inhibitory profile and the pH dependence for the initial linear uptake permeability (PUP) of [(3)H]-E1S suggest that the organic anion transporting polypeptide (OATP) 2B1 is the main transporter involved in the apical E1S PUP in Caco-2 cells from the DSMZ. Furthermore, our results indicate that the efflux transporter breast cancer resistance protein (BCRP) affects E1S PUP, even when uptake is measured at the initial linear uptake phase. Although almost identical results were obtained for cells cultured on plastic dishes and on filter supports, the OATP2B1 stimulator dexamethasone did not affect the PUP for cells grown on dishes but increased [(3)H]-E1S PUP by more than 2-fold for filter grown cells. The basolateral PUP of [(3)H]-E1S of filter grown cells was inhibited by several inhibitors of the bidirectional transporter organic solute transporter α/ß (OSTα/ß). Efflux studies were performed by loading the cells with either [(3)H]-E1S or [(3)H]-taurocholic acid (TCA) and subsequently measuring the efflux of radio labeled substance in the absence or presence of BCRP or OST α/ß inhibitors. Similar effluxes of [(3)H]-E1S was observed across the apical and basolateral membrane, and the apical efflux was greatly decreased in the presence of the BCRP inhibitor fumitremorgin C. In contrast, efflux of [(3)H]-TCA to the basolateral compartment was clearly larger than to the apical compartment. Trans-stimulation of basolateral [(3)H]-E1S efflux was observed in the presence of taurolithocholic acid (TLC), although none of the applied OSTα/ß inhibitors were able to confirm the existence of carrier-mediated efflux at the basolateral membrane, neither for [(3)H]-E1S nor for [(3)H]-TCA. These results highlight the importance of transporter interplay for E1S and drug compounds in Caco-2 cells and emphasize the importance of identifying the basolateral transporters in these cells.

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

Controlled-pH tissue cleanup protocol for signal enhancement of small molecule drugs analyzed by MALDI-MS imaging.

The limit of detection of low-molecular weight compounds in tissue sections, analyzed by matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI), was significantly improved by employing sample washing using a pH-controlled buffer solution. The pH of the washing solutions were set at values whereby the target analytes would have low solubility. Washing the tissue sections in the buffered solution resulted in removal of endogenous soluble ionization-suppressing compounds and salts, while the target compound remained in situ with minor or no delocalization during the buffered washing procedure. Two pharmaceutical compounds (cimetidine and imipramine) and one new protease inhibitor compound were successfully used to evaluate the feasibility of the pH-controlled tissue washing protocol for MALDI-MSI. Enhancement in signal-to-noise ratio was achieved by a factor of up to 10.

The systematic position of Opistocystidae (Annelida, Clitellata) revealed by DNA data.

Opistocystidae Cernosvitov, 1936 is a largely Neotropical oligochaete taxon containing seven species. Its familial status has never been formally challenged, although possible close relationships with Naididae and Phreodrilidae have been noted. Mitochondrial 12S and 16S rDNA, and nuclear 18S rDNA, of a range of aquatic oligochaete taxa, including Trieminentia corderoi (Opistocystidae), were analysed by Bayesian inference. This showed that T. corderoi is a derived lineage within Naididae, closely related to Pristina and its monotypic subfamily Pristininae. Opistocystidae as a whole (with its three genera, Opistocysta, Trieminentia, and Crustipellis) is thus likely to be a group within Naididae.

Optimization of sample preparation for transporter protein quantification in tissues by LC-MS/MS.

BACKGROUND: Reproducible quantification of drug transporter protein expression in tissues is important for predicting transporter mediated drug disposition. Many mass-spectrometry based transporter protein quantification methods result in high variability of the estimated transporter quantities. Therefore, we aimed to evaluate and optimize mass spectrometry-based quantification method for drug transporter proteins in tissues. MATERIALS AND METHODS: Plasma membrane (PM) proteins from mouse tissues were isolated by applying three extraction protocols: commercial plasma membrane extraction kit, tissue homogenization by Potter-Elvehjem homogenizer in combination with sucrose-cushion ultracentrifugation, and PM enrichment with Tween 40. Moreover, five different protein digestion protocols were applied on the same PM fraction. PM isolation and digestion protocols were evaluated by measuring the amount of transporter proteins by liquid chromatography-tandem mass spectrometry in selected reaction monitoring mode. RESULTS: Mouse liver homogenization by Potter-Elvehjem homogenizer in combination with sucrose-cushion ultracentrifugation and PM enrichment with Tween 40 resulted in two times higher transporter protein quantity (Breast cancer resistance protein (Bcrp) 18.0 fmol/µg protein) in comparison with the PM samples isolated by extraction kit (Bcrp 9.8 fmol/µg protein). The evaluation of protein digestion protocols revealed that the most optimal protocol for PM protein digestion is with Lys-C and trypsin, in combination with trypsin enhancer and heat denaturation. Overall, quantities of Bcrp and Na+/K + ATPase proteins evaluated in mouse liver and kidney cortex by using our optimized PM isolation method, as well as, established digestion protocol were two to three times higher than previously reported and coefficient of variation (CV) for technical replicates was below 10%. CONCLUSION: We have established an improved transporter protein quantification methodology by optimizing PM isolation and protein digestion procedures. The optimized procedure resulted in a higher transporter protein yield and improved precision.

An ultrastructural study of the cuticle in the marine annelid Heterodrilus (Tubificidae, Clitellata).

The ultrastructure of the cuticle in four species of the marine Heterodrilus (H. paucifascis, H. pentcheffi, H. flexuosus, H. minisetosus) is investigated with transmission electron microscopy. The noncellular cuticle consists of several parts; closest to the epidermis is a thick zone of collagen fibers embedded in a matrix. The matrix continues outside the fiber zone, forming a layered epicuticle. The external surface of the epicuticle is covered by evenly distributed, membrane-bound bodies, termed epicuticular projections. The epicuticular projections have their longitudinal axis perpendicular to the surface of the cuticle and are attached to the surface by either the surrounding membrane itself or by short pedestals. Microvilli, extensions from the epidermal cells, penetrate and sometimes pass completely through the cuticle. There is interspecific variation in the morphology of the cuticle. The four studied species differ in the arrangement of the collagen fibers, from irregularly distributed fibril bundles to orthogonally arranged fiber layers, as well as in the number and density of layers in the epicuticle. One of the studied species, H. paucifascis, shows intraspecific variation, which is associated with sample locality. The Bahamian specimens of H. paucifascis have four layers in the epicuticle, club-shaped epicuticular projections, and collagen fibers forming a less defined orthogonal grid, while the Belizean specimens have three layers in the epicuticle, epicuticular projections with a bulging part at midlevel, and a distinct orthogonal grid. Based on these findings the variation in the morphology of the cuticle appears to be dependent on both phylogenetic constraints, and functional and environmental factors.

An optimized automated assay for determination of metabolic stability using hepatocytes: assay validation, variance component analysis, and in vivo relevance.

Screening of new chemical entities for metabolic stability using hepatocytes is routinely used in drug discovery. To enhance compound throughput, an optimized automated microassay for determination of intrinsic clearance was developed. Dulbecco's modified Eagle's medium, Hanks' balanced salt solution, and Leibovitz L-15 medium (L-15) were tested for their ability to maintain cell viability during incubation in 96-well plates. L-15 was found to keep pH within 0.1 units and maintain high viability during several hours of incubation. Moreover, two different thawing protocols for cryopreserved hepatocytes were compared. Protocol 2 resulted in a nearly 100% increase in post-thaw yield, whereas no difference was observed in cell viability. The microassay was validated using human cryopreserved hepatocytes and 19 reference compounds covering the most important phase I and II liver metabolizing enzymes ranging from low to medium and high clearance compounds. The day-to-day variation was determined, revealing an overall good precision of the assay. In vitro-in vivo correlations, for both fresh rat and cryopreserved human hepatocytes, were calculated. For 86% (human) and 77% (rat) of the compounds, calculated hepatic clearance was within twofold observed clearance in vivo. Using the validation data, variance component analysis was applied to determine within and between-experiment variability, enabling estimation of variation and detection limit for any combination of repeated experiments and replicate samples. Based on the precision desired, this provides a tool to select the most optimal and cost-effective assay approach for different compounds considering the actual phase in the drug discovery program.

Overexpression of Functional SLC6A3 in Clear Cell Renal Cell Carcinoma.

Purpose: Renal cell carcinoma (RCC) is derived from a tissue with a remarkable capacity for vectorial transport. We therefore performed an unbiased exploration of transporter proteins in normal kidney and kidney cancer to discover novel clinical targets.Experimental Design: Using The Cancer Genome Atlas (TCGA) database, we investigated differences in membrane transporter expression in clear cell RCC (ccRCC) and normal kidney. We identified the dopamine transporter SLC6A3 as a specific biomarker for ccRCC. To investigate the functionality of SLC6A3, we used a [3H]-dopamine uptake assay on ccRCC cells. We further explored the effect of hypoxia-inducible factor (HIF) proteins on SLC6A3 expression by introducing siRNA in ccRCC cells and by hypoxic treatment of nonmalignant cells.Results: We show that ccRCC expresses very high transcript levels of SLC6A3 in contrast to normal kidney tissue and other tumor types, which do not express appreciable levels of this transporter. Importantly, we demonstrate that the elevated expression of SLC6A3 in ccRCC cells is associated with specific uptake of dopamine. By targeting the expression of HIF-1α and HIF-2α, we could show that SLC6A3 expression is primarily influenced by HIF-2α and that hypoxia can induce SLC6A3 expression in normal renal cells.Conclusions: We conclude that the dopamine transporter SLC6A3 constitutes a novel biomarker that is highly specific for ccRCC. We further postulate that the protein can be exploited for diagnostic or therapeutic purposes for detection or treatment of ccRCC. Clin Cancer Res; 23(8); 2105-15. ©2016 AACR.

Sight or smell? Behavioural and heart rate responses in subordinate rainbow trout exposed to cues from dominant fish.

Many animals, including fish, can utilize both vision and the chemical senses in intra-specific communication. However, the relative influence of these sensory modalities on behavioral and physiological responses in social interactions is not well understood. The aim of this study was therefore to investigate the relative effects of visual and chemical stimuli from dominant individuals on the behavioral and physiological responses of subordinate rainbow trout (Oncorhynchus mykiss). External electrodes were used to detect ECG signals from free-swimming fish. This method allowed the simultaneous recording of behavioral and physiological responses, and possible sex differences in these responses were also investigated. The results suggest that, in this context, visual cues are more important than chemical cues in settling the social hierarchy in rainbow trout because a combination of chemical and visual exposure generally yielded a response in focal fish that was similar to the response elicited by visual exposure alone. Both activity and physiological responses were most pronounced during the first ten seconds after exposure, with subordinate fish moving closer to the dominant, accompanied by a strong bradycardic response. Furthermore, females acted more boldly and moved closer to the dominant fish than males, but here the effect of the modes was additive, with a stronger effect of the combined visual and chemical exposure. Overall, the extra information furnished to the fish in the form of chemical cues did not change either the behavioral or the physiological response. This result suggests that visual cues are more important than chemically mediated ones for social communication and individual recognition in rainbow trout.

Drug compound characterization by mass spectrometry imaging in cancer tissue.

MALDI mass spectrometry imaging (MSI) provides a technology platform that allows the accurate visualization of unlabeled small molecules within the two-dimensional spaces of tissue samples. MSI has proven to be a powerful tool-box concept in the development of new drugs. MSI allows unlabeled drug compounds and drug metabolites to be detected and identified and quantified according to their mass-to-charge ratios (m/z) at high resolution in complex tissue environments. Such drug characterization in situ, by both spatial and temporal behaviors within tissue compartments, provide new understandings of the dynamic processes impacting drug uptake and metabolism at the local sites targeted by therapy. Further, MSI in combination with histology and immunohistochemistry, provides the added value of defining the context of cell biology present at the sites of drug localization thus providing invaluable information relating to treatment efficacy. In this report we provide mass spectrometry imaging data within various cancers such as malignant melanoma in patients administered with vemurafenib, a protein kinase inhibitor that is targeting BRAF mutated proteins and that has shown significant efficacy in restraining disease progression. We also provide an overview of other examples of the new generation of targeted drugs, and demonstrate the data on personalized medicine drugs localization within tumor compartments within in vivo models. In these cancer models we provide detailed data on drug and target protein co-localization of YCG185 and sunitinib. These drugs are targeting VEGFR2 within the angiogenesis mechanism. Our ability to resolve drug uptake at targeted sites of directed therapy provides important opportunities for increasing our understanding about the mode of action of drug activity within the environment of disease.

Effect of calmodulin antagonists on phospholipase D activity in SH-SY5Y cells.

The aim of this study was to investigate the involvement of calmodulin in phospholipase D activation in SH-SY5Y cells. Cells prelabelled with [3H]-palmitic acid were incubated with calmodulin antagonists and/or other compounds. Phosphatidylethanol, a specific marker for phospholipase D activity, and phosphatidic acid were analysed. The calmodulin antagonists, calmidazolium and trifluoperazine, induced an extensive increase in phosphatidylethanol formation, and thus increased basal phospholipase D activity, in a dose- and time-dependent manner. The effect of calmidazolium on carbachol-induced activation of muscarinic receptors was also studied. Calmidazolium did not significantly affect the amount of phosphatidylethanol formed following carbachol addition. However, taking into account the increase in basal activity observed after calmidazolium addition, calmidazolium probably inhibits the muscarinic receptor-induced phospholipase D activation. In addition to phosphatidylethanol, basal phosphatidic acid levels were also increased after calmidazolium and trifluoperazine addition. Incubation with calmidazolium (10 microM) for 10 min induced a two-fold increase in phosphatidic acid. The calmidazolium-induced increase in basal phospholipase D activity was not affected by the protein kinase inhibitors H7 and staurosporine. On the other hand tyrosine kinase inhibitors abolished the calmidazolium-induced activation of phospholipase D. Calmidazolium also induced tyrosine phosphorylation in parallel to the phospholipase D activation. In conclusion, our data indicate that calmodulin antagonists induce phospholipase D activity in SH-SY5Y cells via a tyrosine kinase dependent pathway. This may point to a negative control of phospholipase D by calmodulin although a calmodulin-independent mechanism cannot be excluded. Calmodulin antagonists may be useful tools to further elucidate the mechanisms of phospholipase D regulation.

Development of the genital ducts in Telmatodrilinae (Tubificidae, Clitellata).

In Tubificidae, the male genital duct comprises a funnel in the testes segment, followed by a vas deferens, an atrium, and, frequently, a copulatory structure in the adjacent ovarian segment. There may also be a diffuse or compact prostate gland in association with the duct. The morphology and position of the genital ducts are important for the classification of the oligochaetous Clitellata. Different parts of the male duct, however, have been named without regard to whether they are homologous or not. One way to establish better hypotheses of homology is to study the detailed morphology and/or the development of the genital ducts. The morphogenesis of the genital ducts in Alexandrovia onegensis (Telmatodrilinae) is described. The male funnel originates by multiplication of peritoneal (mesodermal) cells in the posterior septum in the testes segment. A cord of these cells breaks through the septum and grows backwards into the next segment, where it connects to the epidermis. This cord gives rise to the vas deferens, and is therefore mesodermal in origin. The atrium in A. onegensis develops from a primary epidermal (ectodermal) invagination. The vas deferens and atrium connect and a continuous duct from the testes segment to the exterior is formed. Several compact prostate glands develop along the atrium, each being formed from cells in the atrial epithelium. The spermatheca develops from an invagination of the epidermis in the testes segment. The female duct is formed from peritoneal (mesodermal) cells in the posterior septum in the ovarian segment. These developmental findings strengthen the hypothesis about a closer relationship between the Telmatodrilinae and Tubificinae (both Tubificidae).

Gene expression analysis of membrane transporters and drug-metabolizing enzymes in the lung of healthy and COPD subjects.

This study describes for the first time the expression levels of genes encoding membrane transporters and drug-metabolizing enzymes in the lungs of ex-smoking patients with chronic obstructive pulmonary disease (COPD). Membrane transporters and drug-metabolizing enzymes are key determinants of drug uptake, metabolism, and elimination for systemically administered as well as inhaled drugs, with consequent influence on clinical efficacy and patient safety. In this study, while no difference in gene expression was found between healthy and COPD subjects, we identified a significant regional difference in mRNA expression of both membrane transporters and drug-metabolizing enzymes between central and peripheral tissue in both healthy and COPD subjects. The majority of the differentially expressed genes were higher expressed in the central airways such as the transporters SLC2A1 (GLUT1), SLC28A3 (CNT3), and SLC22A4 (OCTN1) and the drug-metabolizing enzymes GSTZ1, GSTO2, and CYP2F1. Together, this increased knowledge of local pharmacokinetics in diseased and normal lung may improve modeling of clinical outcomes of new chemical entities intended for inhalation therapy delivered to COPD patients. In addition, based on the similarities between COPD and healthy subjects regarding gene expression of membrane transporters and drug-metabolizing enzymes, our results suggest that clinical pharmacological studies in healthy volunteers could be a valid model of COPD patients regarding drug disposition of inhaled drugs in terms of drug metabolism and drug transporters.

Identification of novel substrates and structure-activity relationship of cellular uptake mediated by human organic cation transporters 1 and 2.

Recently the clinical importance of human organic cation transporters 1 (hOCT1/SLC22A1) and 2 (hOCT2/SLC22A2) in drug disposition, for example, clearance, toxicity, and drug-drug interactions, have been highlighted [Annu. Rev. Pharmacol. Toxicol. 2012, 52, 249-273; Nat. Rev. Drug Discovery 2010, 9 (3), 215-236]. Consequently, there is an extensive need for experimental assessment of structure-transport relationships as well as tools to predict drug uptake by these transporters in ADMET (absorption, distribution, metabolism, excretion, toxicity) investigations. In the present study, we developed a robust assay for screening unlabeled compound uptake by hOCT1 and hOCT2 using transfected HEK293 cells. For the first time, an extensive data set comprising uptake of 354 compounds is presented. As expected, there was a large overlap in substrate specificity between the two organic cation transporters. However, several compounds selectively taken up by either hOCT1 or hOCT2 were identified. In particular, a chemical series of phenylthiophenecarboxamide ureas was identified as selective hOCT1 substrates. Moreover, the drivers for transport differed: molecular volume was the most important determinant of hOCT1 substrates, whereas H-bonding parameters like polar surface area (PSA) dominated for hOCT2.

Effect of cryopreservation on the activity of OATP1B1/3 and OCT1 in isolated human hepatocytes.

Drug metabolism in liver is the major pathway for xenobiotic elimination from the body. Access to intracellular metabolising enzymes is possible through passive diffusion of lipophilic drugs through cell membrane or active uptake of more polar drugs by specific uptake transporters. Organic Anion Transporting Polypeptides (OATP/SLCO) and Organic Cation Transporters (OCT/SLC22A) are among the most important transporters involved in xenobiotic transport into hepatocytes. Isolated hepatocytes are the model of choice for drug metabolism and drug transport investigations. These primary cells are used either as fresh directly after isolation from liver biopsies, or after subsequent cryopreservation in liquid nitrogen. While cryopreserved hepatocytes are a more convenient and flexible tool for in vitro investigations, information on the functionality of transporter activity after cryopreservation is still sparse. The present study investigated the effect of cryopreservation of human hepatocytes on the uptake of [(3)H]-estradiol-17ß-glucuronide (E(2)17ßG, substrate of OATP1B1/3/SLCO1B1/3) and [(3)H]-1-methyl-4-phenylpyridinium (MPP+, substrate of OCT1/SLC22A1) into hepatocytes from 6 and 5 human donors, respectively. The results showed that cryopreserved human hepatocytes display carrier-mediated uptake of E(2)17ßG and MPP+. While the affinity of E(2)17ßG for OATP1B1/3/SLCO1B1/3 was not affected by cryopreservation (Km unchanged, the Wilcoxon signed pair t test gave p=1), V(max) and CL(uptake) values decreased in average by 47% (p=0.06). The passive diffusion of E(2)17ßG decreased significantly after cryopreservation (p=0.03). Cryopreservation did not affect Km, V(max) or the passive diffusion of MPP+ in human hepatocytes. In conclusion, the present study showed that cryopreserved human hepatocytes are useful tool to investigate hepatic uptake mediated by OATP1B1/3/SLCO1B1/3 or OCT1/SLC22A1, two of the most important hepatic uptake transporters.

Challenges in inhaled product development and opportunities for open innovation.

Dosimetry, safety and the efficacy of drugs in the lungs are critical factors in the development of inhaled medicines. This article considers the challenges in each of these areas with reference to current industry practices for developing inhaled products, and suggests collaborative scientific approaches to address these challenges. The portfolio of molecules requiring delivery by inhalation has expanded rapidly to include novel drugs for lung disease, combination therapies, biopharmaceuticals and candidates for systemic delivery via the lung. For these drugs to be developed as inhaled medicines, a better understanding of their fate in the lungs and how this might be modified is required. Harmonized approaches based on 'best practice' are advocated for dosimetry and safety studies; this would provide coherent data to help product developers and regulatory agencies differentiate new inhaled drug products. To date, there are limited reports describing full temporal relationships between pharmacokinetic (PK) and pharmacodynamic (PD) measurements. A better understanding of pulmonary PK and PK/PD relationships would help mitigate the risk of not engaging successfully or persistently with the drug target as well as identifying the potential for drug accumulation in the lung or excessive systemic exposure. Recommendations are made for (i) better industry-academia-regulatory co-operation, (ii) sharing of pre-competitive data, and (iii) open innovation through collaborative research in key topics such as lung deposition, drug solubility and dissolution in lung fluid, adaptive responses in safety studies, biomarker development and validation, the role of transporters in pulmonary drug disposition, target localisation within the lung and the determinants of local efficacy following inhaled drug administration.