Temporal expression of transporters and receptors in a rat primary co-culture blood–brain barrier model

Abstract

1.?The more relevant primary co-cultures of brain microvessel endothelial cells and astrocytes (BMEC) are less utilized for screening of potential CNS uptake when compared to intestinal and renal cell lines.

2.?In this study, we characterized the temporal mRNA expression of major CNS transporters and receptors, including the transporter regulators Pxr, Ahr and Car in a rat BMEC co-cultured model. Permeability was compared with the Madin–Darby canine kidney (MDCKII)-MDR1 cell line and rat brain in situ perfusion model.

3.?Our data demonstrated differential changes in expression of individual transporters and receptors over the culture period. Expression of ATP-binding cassette transporters was better retained than that of solute carrier transporters. The insulin receptor (IR) was best maintained among investigated receptors. AhR demonstrated high mRNA expression in rat brain capillaries and expression was better retained than Pxr or Car in culture. Mdr1b expression was up-regulated during primary culture, albeit Mdr1a mRNA levels were much higher. P-gp and Bcrp-1 were highly expressed and functional in this in vitro system.

4.?Permeability measurements with 18 CNS marketed drugs demonstrated weak correlation between rBMEC model and rat in situ permeability and moderate correlation with MDCKII-MDR1 cells.

5.?We have provided appropriate methodologies, as well as detailed and quantitative characterization data to facilitate improved understanding and rational use of this in vitro rat BBB model.     

Cholesterol and non-cholesterol sterol transporters: ABCG5, ABCG8 and NPC1L1: a review

1.?Whole-body sterol (cholesterol and xenosterol) balance is delicately regulated by the gastrointestinal tract and liver, which control sterol absorption and excretion, respectively, in addition to the contribution to the cholesterol pool by whole-body cholesterol synthesis. In the past ten years enormous strides have been made not only in establishing that specific transporters mediate the entry and exit of sterols and how these may regulate selective sterol access to the body pools, but also in how these pathways operate to integrate these physiological pathways.

2.?The entry of sterols from the gastrointestinal and biliary canalicular lumen into the body is mediated by NPC1L1, which was discovered by a novel method, via a genomics–bioinformatics approach.

3.?Identification of the genetic basis responsible for causing sitosterolaemia, characterized by plant sterol accumulation, led to the identification of two half-transporters (ABCG5 and ABCG8) that normally efflux plant sterols (and cholesterol) into the intestinal and biliary lumen for faecal excretion.

4.?The objective of this review is to provide up-to-date knowledge on genomics, proteomics and function of these two transporter systems.     

Intestinal absorption of pallidifloside D are limited by P-glycoprotein in mice

1.?Pallidifloside D, a saponin glycoside constituent from the total saponins of Smilax riparia, had been proved to be very effective in hyperuricemic control. But it is poorly bioavailable after oral administration. Here, we determined the role of P-glycoprotein (P-gp) in the intestinal absorption of Pallidifloside D.

2.?We found that Pallidifloside D significantly stimulated P-gp ATPase activity in vitro ATPase assay with a small EC₅₀ value of 0.46?μM.

3.?In the single-pass perfused mouse intestine model, the absorption of Pallidifloside D was not favored in the small intestine (duodenum, jejunum and ileum) with a P*_w value of 0.35–0.78. By contrast, this compound was well-absorbed in the colon with a P*_w value of 1.23. The P-gp inhibitors cyclosporine significantly enhanced Pallidifloside D absorption in all four intestinal segments (duodenum, jejunum, ileum and colon) and the fold change ranged from 5.5 to 15.3. Pharmacokinetic study revealed that cyclosporine increased the systemic exposure of Pallidifloside D by a 2.5-fold after oral administration.

4.?These results suggest that P-gp-mediated efflux is a limiting factor for intestinal absorption of Pallidifloside D in mice.     

Peptide transporters and their roles in physiological processes and drug disposition

1.?The peptide transporters belong to the peptide transporter (PTR) family and serve as integral membrane proteins for the cellular uptake of di- and tripeptides in the organism. By their ability also to transport peptidomimetics and other substrates with therapeutic activities or precursors of pharmacologically active agents, they are of considerable importance in pharmacology.

2.?PEPT1 is the low-affinity, high-capacity transporter and is mainly expressed in the small intestine, whereas PEPT2 is the high-affinity, low-capacity transporter and has a broader distribution in the organism.

3.?Targeted mouse models have revealed PEPT2 to be the dominant transporter for the reabsorption of di- and tripeptides and its pharmacological substrates in the organism, and for the removal of these substrates from the cerebrospinal fluid. Moreover, the peptide transporters undergo physiological and pharmacological regulation and, of great interest, are present in disease states where PEPT1 exhibits ectopic expression in colonic inflammation.

4.?The paper reviews the structural characteristics of the peptide transporters, the structural requirements for substrates, the distribution of the peptide transporters in the organism, and finally their regulation in the organism in healthy and pathological situations.     

Characterization of gastrointestinal absorption of digoxin involving influx and efflux transporter in rats: application of mdr1a knockout (−/−) rats into absorption study of multiple transporter substrate

Abstract

1.?This study was aimed to characterize gastrointestinal absorption of digoxin using wild-type (WT) and multidrug resistance protein 1a [mdr1a; P-glycoprotein (P-gp)] knockout (?/?) rats.

2.?In WT rats, the area under the plasma concentration–time curve (AUC) of oral digoxin increased after oral pretreatment with quinidine at 30?mg/kg compared with non-treatment, but the increasing ratio tended to decrease at a high dose of 100?mg/kg. In mdr1a (?/?) rats, however, quinidine pretreatment caused a dose-dependent decrease in the AUC.

3.?Quinidine pretreatment did not alter the hepatic availability of digoxin, indicating that the changes in the digoxin AUC were attributable to inhibition of the absorption process by quinidine; i.e. inhibition of influx by quinidine in mdr1a (?/?) rats and inhibition of efflux and influx by quinidine in WT rats.

4.?An in situ rat intestinal closed loop study using naringin implied that organic anion transporting peptide (Oatp) 1a5 may be a responsible transporter in the absorption of digoxin.

5.?These findings imply that the rat absorption behavior of digoxin is possibly governed by Oatp1a5-mediated influx and P-gp-mediated efflux. The mdr1a (?/?) rat is therefore a useful in vivo tool to investigate drug absorption associated with multiple transporters including P-gp.     

The regulation of human hepatic drug transporter expression by activation of xenobiotic-sensing nuclear receptors

Introduction: If a drug is found to be an inducer of hepatic drug metabolizing enzymes via activation of nuclear receptors such as pregnane X receptor (PXR) or constitutive androstane receptor (CAR), it is likely that drug transporters regulated through these same receptors will be induced as well. This review highlights what is currently known about the molecular mechanisms that regulate transporter expression and where the research is directed.

Areas covered: This review is focused on publications that describe the role of activated hepatic nuclear receptors in the subsequent regulation of drug uptake and/or efflux transporters following exposure to xenobiotics.

Expert opinion: Many of the published studies on the role of nuclear receptors in the regulation of drug transporters involve non-human test animals. But due to species response differences, these associations are not always applicable to humans. For this reason, some relevant human in vitro models have been developed, such as primary or cryopreserved human hepatocytes, human liver slices, or HepG2 or HuH7 cell lines transiently or stably transfected with PXR expression and reporter constructs as well as in vivo models such as PXR-humanized mice. These human-relevant test systems will continue to be developed and applied for the testing of investigational drugs.     

In vitro drug–drug interactions of budesonide: inhibition and induction of transporters and cytochrome P450 enzymes

1.?Budesonide is a glucocorticoid used in the treatment of several respiratory and gastrointestinal inflammatory diseases. Glucocorticoids have been demonstrated to induce cytochrome P450 (CYP) 3A and the efflux transporter P-glycoprotein (P-gp). This study aimed to evaluate the potential of budesonide to act as a perpetrator or a victim of transporter- or CYP-mediated drug–drug interactions (DDIs).

2.?In vitro studies were conducted for P-gp, breast cancer resistance protein and organic anion and cation transporters (OATP1B1, OATP1B3, OAT1, OAT3, OCT2) in transporter-transfected cells. Changes in mRNA expression in human hepatocytes and enzyme activity in human liver microsomes by budesonide were determined for CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A.

3.?The data indicated that budesonide is a substrate of P-gp but is not a substrate or an inhibitor of the other transporters investigated. Budesonide is neither an inducer nor an inhibitor of major CYP enzymes. The effect of P-gp on budesonide disposition is anticipated to be low owing to CYP3A-mediated clearance.

4.?Collectively, our data indicate there is a low risk of budesonide perpetrating clinical DDIs mediated by the transporters or CYPs studied.     

In vitro evaluation of potential drug interactions mediated by cytochrome P450 and transporters for luseogliflozin,an SGLT2 inhibitor

1.?We evaluated potential in vitro drug interactions of luseogliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, mediated by CYP inhibition, CYP induction and drug transporters using human liver microsomes, primary hepatocytes and recombinant cells-expressing efflux or uptake transporters, respectively.

2.?Human CYP inhibition studies indicated that luseogliflozin was a weak inhibitor for CYP2C19 with an IC₅₀ value of 58.3?μM, whereas it was not an inhibitor of the other eight major isoforms that were tested. The exposure of primary hepatocytes to luseogliflozin for 72?hrs weakly induced CYP3A4 at a concentration of 10?μM, whereas it did not induce CYP1A2 or CYP2B6 at concentrations of 0.1–10?μM.

3.?An in vitro transport study suggested that luseogliflozin is a substrate for human P-glycoprotein (P-gp), but not for breast cancer resistance protein (BCRP), organic anion transporting polypeptide (OATP) 1B1 and OATP1B3, organic anion transporter (OAT) 1 and OAT3, or organic cation transporter (OCT) 2. Luseogliflozin weakly inhibited OATP1B3 with an IC₅₀ value of 93.1?μM, but those for other transporters are greater than 100?μM.

4.?Based on the therapeutic plasma concentration of the drug, clinically relevant drug interactions are unlikely to occur between luseogliflozin and coadministered drugs mediated by CYPs and/or transporters.     

Interplay of metabolizing enzymes and transporter of xenobiotics

1.?Xenobiotics are metabolized and eliminated through the coordinated interplay of their metabolizing enzymes and transporters. However, these two activities in vitro are measured separately, with the addition of ATP as a pre-requisite.

2.?We propose a human renal cell-line model which integrates the sulfate and glutathione conjugation of xenobiotics with the efflux of their respective conjugates. Sulfation and glutathionylation represent two major Phase II detoxification of xenobiotics in man. The reactions are catalyzed, respectively, by phenolsulfotransferase and glutathione-S-transferase followed by extrusion of their respective conjugates.

3.?Using Ko-143, a specific inhibitor of breast cancer resistance protein (BCRP), an ATP-binding cassette (ABC) transporter, we identified this transporter to be responsible for the efflux of p-cresol sulfate, harmol sulfate and the glutathione conjugate of 1-chloro-2,4-dinitrobenzene.

4.?The conjugation-cum-efflux was inhibited by oligomycin and uncouplers, which highlights the role of cellular mitochondria in providing ATP for the biosynthesis of their conjugating agents, 3′-phosphoadenosine-5′-phosphosulfate (PAPS) and reduced glutathione as well as for the transport function of BCRP.

5.?The human 786-O renal cell-line provides a “3-in-1” system linking ATP biosynthesis to metabolism of xenobiotics and their ultimate transport and elimination by BCRP; this integrated system was not apparent in other human cell-lines examined.     

Characterization of renal tubular apical efflux of zonampanel,an α-amino-3-hydroxy-5- methylisoxazole-4-propionate receptor antagonist,in humans

1.?Zonampanel, a novel α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor antagonist, is mainly excreted unchanged via renal tubular secretion. The renal apical transport transport of zonampanel was examined in this study using HEK293 cells expressing human organic anion transporter 4 (OAT4/SLC22A11), and membrane vesicles prepared from Sf-9 insect cells expressing human multidrug resistance-associated protein 2 (MRP2/ABCC2), MRP4 (ABCC4), and breast cancer resistance protein (BCRP/ABCG2).

2.?Glutaric acid, a model dicarboxylate, trans-stimulated the uptake of [¹⁴C]zonampanel by OAT4, suggesting that zonampanel was transported by OAT4 via an exchange with dicarboxylate. Considering the endogenous dicarboxylate gradient, OAT4 seems to transport zonampanel in the direction of reabsorption rather than secretion. For MRP2, MRP4, and BCRP, zonampanel selectively inhibited the activity of MRP4 (K_i?=?41.3 µM). Marked transport of [¹⁴C]zonampanel was observed only for MRP4 (K_m?=?33.7 µM).

3.?In conclusion, the data indicate that MRP4 was the apical efflux transporter that contributed to the active renal tubular secretion of zonampanel in humans, in concert with the apical reabsorption transporter OAT4 and basolateral uptake transporters.     

LOSS of Mrp1 alters detoxification enzyme expression in a tissue‐ and hormonal‐status‐specific manner

The multidrug resistance‐associated protein1 (MRP1/ABCC1) is a member of the ABCC transporter subfamily that mediates the efflux of pharmaceuticals, xenobiotics and steroid hormones, typically as glutathione, glucuronide or sulfate conjugates. Since loss of one transporter can be compensated by increasing the expression of other transporters and conjugation enzymes, we sought to examine compensatory changes in phase I, II and III enzyme expression in extrahepatic tissues, including the kidney, lungs and small intestine of intact or castrated Mrp1^?/? male mice. In the kidney, the expression of several P450s, sulfotransferase 1a1 (Sult), glucuronosyltransferases (Ugt) and Mrps2–4, were significantly changed owing to castration alone. The only time genotype mattered was between the castrated FVB and Mrp1 knockout mice. In contrast, expression of the Ugts, Sult 1a1 and Mrp3 in the lungs was significantly downregulated in the Mrp1 knockout mice, so based exclusively on genotype. In the small intestine, there were interactions between steroid hormone levels and genotype, as the expression differences were only found in mice lacking Mrp1, and were changed between intact and castrated animals. The mechanism behind this pattern of expression may be to due to Nrf2 regulation, as its expression mirrors that of the phase II and phase III enzymes. These results indicate that compensatory responses owing to the loss of Mrp1 vary dramatically, depending on the particular tissue. This information will aid in the understanding of how drug uptake, disposition and elimination can be influenced by both hormone status and the presence and magnitude of transporter expression. Copyright © 2012 John Wiley & Sons, Ltd.     

Efflux proteins at the blood–brain barrier: review and bioinformatics analysis

1.?Efflux proteins at the blood–brain barrier provide a mechanism for export of waste products of normal metabolism from the brain and help to maintain brain homeostasis. They also prevent entry into the brain of a wide range of potentially harmful compounds such as drugs and xenobiotics.

2.?Conversely, efflux proteins also hinder delivery of therapeutic drugs to the brain and central nervous system used to treat brain tumours and neurological disorders. For bypassing efflux proteins, a comprehensive understanding of their structures, functions and molecular mechanisms is necessary, along with new strategies and technologies for delivery of drugs across the blood–brain barrier.

3.?We review efflux proteins at the blood–brain barrier, classified as either ATP-binding cassette (ABC) transporters (P-gp, BCRP, MRPs) or solute carrier (SLC) transporters (OATP1A2, OATP1A4, OATP1C1, OATP2B1, OAT3, EAATs, PMAT/hENT4 and MATE1).

4.?This includes information about substrate and inhibitor specificity, structural organisation and mechanism, membrane localisation, regulation of expression and activity, effects of diseases and conditions and the principal technique used for in vivo analysis of efflux protein activity: positron emission tomography (PET).

5.?We also performed analyses of evolutionary relationships, membrane topologies and amino acid compositions of the proteins, and linked these to structure and function.     

Multidrug and toxic compound extrusion (MATE)-type proteins as anchor transporters for the excretion of metabolic waste products and xenobiotics

1.?Multidrug and toxic compound extrusion (MATE)-type transporters, which were first identified as a bacterial drug transporter family, are present in almost all prokaryotes and eukaryotes, and are thus one of the mostly conserved transporter families in nature.

2.?Recently, a mammalian MATE transporter was shown to be a long hypothesized electroneutral H⁺/organic cation exporter that is responsible for the excretion of metabolic waste products and xenobiotics at renal brush border membranes and bile canaliculi. Plant MATE-type transporters are involved in the detoxification of metals and secondary metabolites such as phenols through their vesicular storage or extrusion at the plasma membrane.

3.?Thus, MATE transporters are involved in one of the basic mechanisms that maintain homeostasis through the excretion of metabolic waste products and xenobiotics in nature.     

Absorption characteristic of paeoniflorin-6′-O-benzene sulfonate (CP-25) in in situ single-pass intestinal perfusion in rats

1.?Paeoniflorin-6′-O-benzene sulfonate (CP-25) was synthesized to improve the poor oral absorption of paeoniflorin (Pae).

2.?This study was performed to investigate the absorptive behavior and mechanism of CP-25 in in situ single-pass intestinal perfusion in rats, using Pae as a control.

3.?The results showed that intestinal absorption of CP-25 was neither segmental nor sex dependent. However, the main segment of intestine that absorbed Pae was the duodenum. Furthermore, passive transport was confirmed to be the main absorption pattern of CP-25. More importantly, the absorption of CP-25 was much higher than Pae in the small intestine.

4.?Among the ABC transporter inhibitors, the absorption rate of Pae increased in the presence of P-gp inhibitors verapamil and GF120918, which indicated that Pae was a substrate of P-glycoprotein (P-gp), however, such was not observed in the presence of breast cancer resistance protein and multidrug resistance-associated protein 2. Finally, the ABC transporter inhibitors did not have any significant impact on CP-25 as demonstrated in the parallel studies.

5.?CP-25 could improve the poor absorption of Pae, which may be attributed to both the lipid solubility enhancement and its resistance to P-gp-mediated efflux.