Expression, activity and pharmacokinetic impact of ocular transporters

The eye is protected by several tissues that limit the permeability and entry of potentially harmful substances, but also hamper the delivery of drugs in the treatment of ocular diseases. Active transport across the ocular barriers may affect drug distribution, but the impact of drug transporters on ocular drug delivery is not well known. We have collected and critically reviewed the literature for ocular expression and activity of known drug transporters. The review concentrates on drug transporters that have been functionally characterized in ocular tissues or primary cells and on transporters for which there is available expression data at the protein level. Species differences are highlighted, since these may explain observed inconsistencies in the influence of specific transporters on drug disposition. There is variable evidence about the pharmacokinetic role of transporters in ocular tissues. The strongest evidence for the role of active transport is available for the blood-retinal barrier. We explored the role of active transport in the cornea and blood retinal barrier with pharmacokinetic simulations. The simulations show that the active transport is important only in the case of specific parameter combinations. (C) 2017 Elsevier B.V. All rights reserved.Peer reviewe

Efflux Protein Expression in Human Stem Cell-Derived Retinal Pigment Epithelial Cells

Retinal pigment epithelial (RPE) cells in the back of the eye nourish photoreceptor cells and form a selective barrier that influences drug transport from the blood to the photoreceptor cells. At the molecular level, ATP-dependent efflux transporters have a major role in drug delivery in human RPE. In this study, we assessed the relative expression of several ATP-dependent efflux transporter genes (MRP1, -2, -3, -4, -5, -6, p-gp, and BCRP), the protein expression and localization of MRP1, MRP4, and MRP5, and the functionality of MRP1 efflux pumps at different maturation stages of undifferentiated human embryonic stem cells (hESC) and RPE derived from the hESC (hESC-RPE). Our findings revealed that the gene expression of ATP-dependent efflux transporters MRP1, -3, -4, -5, and p-gp fluctuated during hESC-RPE maturation from undifferentiated hESC to fusiform, epithelioid, and finally to cobblestone hESC-RPE. Epithelioid hESC-RPE had the highest expression of MRP1, -3, -4, and P-gp, whereas the most mature cobblestone hESC-RPE had the highest expression of MRP5 and MRP6. These findings indicate that a similar efflux protein profile is shared between hESC-RPE and the human RPE cell line, ARPE-19, and suggest that hESC-RPE cells are suitable in vitro RPE models for drug transport studies. Embryonic stem cell model might provide a novel tool to study retinal cell differentiation, mechanisms of RPE -derived diseases, drug testing and targeted drug therapy

Excess protein enabled dog domestication during severe Ice Age winters

International audienceDogs (Canis familiaris) are the first animals to be domesticated by humans and the only ones domesticated by mobile hunter-gatherers. Wolves and humans were both persistent, pack hunters of large prey. They were species competing over resources in partially overlapping ecological niches and capable of killing each other. How could humans possibly have domesticated a competitive species? Here we present a new hypothesis based on food/resource partitioning between humans and incipient domesticated wolves/dogs. Humans are not fully adapted to a carnivorous diet; human consumption of meat is limited by the liver’s capacity to metabolize protein. Contrary to humans, wolves can thrive on lean meat for months. We present here data showing that all the Pleistocene archeological sites with dog or incipient dog remains are from areas that were analogous to subarctic and arctic environments. Our calculations show that during harsh winters, when game is lean and devoid of fat, Late Pleistocene hunters-gatherers in Eurasia would have a surplus of animal derived protein that could have been shared with incipient dogs. Our partitioning theory explains how competition may have been ameliorated during the initial phase of dog domestication. Following this initial period, incipient dogs would have become docile, being utilized in a multitude of ways such as hunting companions, beasts of burden and guards as well as going through many similar evolutionary changes as humans

Morphology and gene expression of hESC on different maturation stages.

<p>Brightfield micrographs of cell cultures showing the representative morphology of <b>A</b>) undifferentiated hESC (Regea08/017), <b>B</b>) fusiform hESC-RPE, <b>C</b>) epithelioid hESC-RPE, <b>D</b>) cobblestone hESC-RPE. Scale bars, 100 µm, <b>E</b>) Gene expression of <b>1</b>: D407, <b>3</b>: ARPE-19, <b>5</b>: undifferentiated hESC, <b>7</b>: fusiform hESC-RPE, <b>9</b>: epithelioid hESC-RPE, <b>11</b>: cobblestone hESC-RPE, <b>13</b>: hFF. –RT- negative controls (i.e., samples not treated with reverse transcriptase) are placed adjacent to each sample in the same order: <b>2</b>: D407, <b>4</b>: ARPE-19, <b>6</b>: undifferentiated hESC, <b>8</b>: fusiform hESC-RPE, <b>10</b>: epithelioid hESC-RPE, <b>12</b>: cobblestone hESC-RPE, <b>14</b>: hFF. <b>F</b>) Culture periods of the studied samples in all analyses. Cells were selected based on their morphology rather than the culture period.</p

Reverse-transcriptase–PCR primer sequences and used annealing temperatures.

<p>Reverse-transcriptase–PCR primer sequences and used annealing temperatures.</p

Localization of ATP-dependent efflux transporter proteins. A-L

<p>) Confocal micrographs after indirect immunofluorescence labeling with efflux pump proteins MRP-1, -4, or -5 (green), and eye-specific proteins MITF and cellular retinaldehyde-binding protein (CRALBP, both red), the polarization marker Na⁺/K⁺ ATPase (red), and the nuclear label 4′,6′-diamidino-2-phenylidole (blue). In figures <b>M-P</b>) the brightfield micrographs show the same ARPE-19 cells and fusiform, early cobblestone, and cobblestone hESC-RPE as shown in the confocal images. Scale bars, 10 µm.</p

Functional testing of ATP-dependent efflux transporter proteins and viability of cultured cells. A

<p>) Calcein retention in ARPE-19, undifferentiated hESC, fusiform, and cobblestone hESC-RPE, and hFF cells in the presence or absence ( =  control) of efflux protein inhibitors. Retention is expressed as a percentage of fluorescence relative to control (control = 100%). The studies were repeated at least three times for ARPE-19 and fusiform hESC-RPE, and once each for undifferentiated hESC, cobblestone hESC-RPE, and hFF. Data are expressed as mean±SD, *p<0.05, **p<0.01, ***p<0.001. B-E) Representative images of viable (green fluorescence) and dead (red fluorescence) ARPE19 (B,C) and fusiform hESC RPE cells (D,E). The scale bar 100 µM.</p