MDCKII Cells Research Articles

Study on the Effect of Pharmaceutical Excipient PEG400 on the Pharmacokinetics of Baicalin in Cells Based on MRP2, MRP3, and BCRP Efflux Transporters.

Pharmaceutical excipient PEG400 is a common component of traditional Chinese medicine compound preparations. Studies have demonstrated that pharmaceutical excipients can directly or indirectly influence the disposition process of active drugs in vivo, thereby affecting the bioavailability of drugs. In order to reveal the pharmacokinetic effect of PEG400 on baicalin in hepatocytes and its mechanism, the present study first started with the effect of PEG400 on the metabolic disposition of baicalin at the hepatocyte level, and then the effect of PEG400 on the protein expression of baicalin-related transporters (BCRP, MRP2, and MRP3) was investigated by using western blot; the effect of MDCKII-BCRP, MDCKII-BCRP, MRP2, and MRP3 was investigated by using MDCKII-BCRP, MDCKII-MRP2, and MDCKII-MRP3 cell monolayer models, and membrane vesicles overexpressing specific transporter proteins (BCRP, MRP2, and MRP3), combined with the exocytosis of transporter-specific inhibitors, were used to study the effects of PEG400 on the transporters in order to explore the possible mechanisms of its action. The results demonstrated that PEG400 significantly influenced the concentration of baicalin in hepatocytes, and the AUC0-t of baicalin increased from 75.96 ± 2.57 μg·h/mL to 106.94 ± 2.22 μg·h/mL, 111.97 ± 3.98 μg·h/mL, and 130.42 ± 5.26 μg·h/mL (p ˂ 0.05). Furthermore, the efflux rate of baicalin was significantly reduced in the vesicular transport assay and the MDCKII cell model transport assay, which indicated that PEG400 had a significant inhibitory effect on the corresponding transporters. In conclusion, PEG400 can improve the bioavailability of baicalin to some extent by affecting the efflux transporters and thus the metabolic disposition of baicalin in the liver.

Applicability of MDR1 Overexpressing Abcb1KO-MDCKII Cell Lines for Investigating In Vitro Species Differences and Brain Penetration Prediction.

Implementing the 3R initiative to reduce animal experiments in brain penetration prediction for CNS-targeting drugs requires more predictive in vitro and in silico models. However, animal studies are still indispensable to obtaining brain concentration and determining the prediction performance of in vitro models. To reveal species differences and provide reliable data for IVIVE, in vitro models are required. Systems overexpressing MDR1 and BCRP are widely used to predict BBB penetration, highlighting the impact of the in vitro system on predictive performance. In this study, endogenous Abcb1 knock-out MDCKII cells overexpressing MDR1 of human, mouse, rat or cynomolgus monkey origin were used. Good correlations between ERs of 83 drugs determined in each cell line suggest limited species specificities. All cell lines differentiated CNS-penetrating compounds based on ERs with high efficiency and sensitivity. The correlation between in vivo and predicted Kp,uu,brain was the highest using total ER of human MDR1 and BCRP and optimized scaling factors. MDR1 interactors were tested on all MDR1 orthologs using digoxin and quinidine as substrates. We found several examples of inhibition dependent on either substrate or transporter abundance. In summary, this assay system has the potential for early-stage brain penetration screening. IC50 comparison between orthologs is complex; correlation with transporter abundance data is not necessarily proportional and requires the understanding of modes of transporter inhibition.

Aluminum and ABC transporter activity

Aluminum is the third most common element on Earth´s crust and despite its wide use in our workaday life it has been associated with several health risks after overexposure. In the present study the impact of aluminum salts upon ABC transporter activity was studied in the P-GP-expressing human blood-brain barrier cell line hCMEC/D3, in MDCKII cells overexpressing BCRP and MRP2, respectively, and in freshly isolated, functionally intact kidney tubules from Atlantic killifish (Fundulus heteroclitus), which express the analog ABC transporters, P-gp, Bcrp and Mrp2. In contrast to previous findings with heavy metals salts (cadmium(II) chloride or mercury(II) chloride), which have a strong inhibitory effect on ABC transporter activity, or zinc(II) chloride and sodium arsenite, which have a stimulatory effect upon ABC transport function, the results indicate no modulatory effect of aluminum salts on the efflux activity of the human ABC transporters P-GP, BCRP and MRP2 nor on the analog transporters P-gp, Bcrp and Mrp2.

ABCG2 Transports the Flukicide Nitroxynil and Affects Its Biodistribution and Secretion into Milk.

The ABCG2 transporter plays a key role in pharmacological and toxicological processes, affecting bioavailability, tissue accumulation and milk secretion of its substrates. This protein is expressed in several biological barriers acting as a protective mechanism against xenobiotic exposure by pumping out a broad range of compounds. However, its induced expression during lactation in alveolar cells of mammary gland represents a relevant route for active transport of unwanted chemicals into milk. This work aimed to characterize the involvement of ABCG2 in systemic exposure and milk secretion of the flukicide nitroxynil. Using MDCK-II cells overexpressing the transporter, we showed that nitroxynil is an in vitro substrate of different species variants of ABCG2. Moreover, using wild-type and Abcg2-/- mice, we showed that murine Abcg2 clearly affects plasma levels of nitroxynil. We also reported differences in nitroxynil accumulation in several tissues, with almost 2-fold higher concentration in kidney, small intestine and testis of Abcg2-/- mice. Finally, we proved that nitroxynil secretion into milk was also affected by Abcg2, with a 1.9-fold higher milk concentration in wild-type compared with Abcg2-/- mice. We conclude that ABCG2 significantly impacts nitroxynil biodistribution by regulating its passage across biological barriers.

A technological comparison of freeze-dried poly-ɛ-caprolactone (PCL) and poly (lactic-co-glycolic acid) (PLGA) nanoparticles loaded with clozapine for nose-to-brain delivery

Clozapine (CZP) is an atypical antipsychotic, the most effective for the treatment of resistant schizophrenia. With the aim of overcome the limitations of current dosage forms (tablets or suspensions) and routes of administration (oral and intramuscular), this research project proposes nanocarrier systems for intranasal (IN) administration of CZP. In particular, the systems have been developed for the direct nose-to-brain (N2B) route, which allows the direct delivery of the drug to the central nervous system (CNS), bypassing the blood-brain barrier (BBB). This study focusses on the development and investigation of two distinct types of lyophilized polymeric nanoparticles (NPs) consisting of poly- ɛ -caprolactone (PCL) or poly (lactic-co-glycolic acid) (PLGA). The lyophilized formulations were cryoprotected with 5 % (w/v) d-maltose for PLGA-NPs and 25 % (w/v) d-maltose for PCL-NPs. The NPs showed a size of approximately 200 nm, suitable for reuptake from the olfactory nerve to reach the brain directly after the IN administration. In vitro release studies performed in artificial cerebrospinal fluid (aCSF) and simulated nasal fluid (SNF) showed that PLGA NPs released 100 % of the drug in 4 days in SNF, while in aCSF they released about 80 % in 20 days. On the contrary, PCL-NPs released the entire amount of drug within 24 h in both fluids. Furthermore, preliminary in vitro mucoadhesion studies were performed on NPs in liquid form (suspensions) to compare the possible variation in their mucoadhesive properties with respect to powdered NPs, which were tested directly on pig nasal mucosa tissue, where permeability was also assessed. CZP-loaded NPs showed some interactions with mucin in vitro, but no ex-vivo mucoadhesiveness was demonstrated. Furthermore, the results of permeation indicated that the use of CZP-PCL-NPs did not increase the effective permeation of CZP into the mucosa; however, a faster onset of permeation compared to free CZP and a controlled profile were demonstrated. Toxicity studies on primary human olfactory mucosa cells extracted from volunteer patients and on MDCKII cells were also done. Finally, stability studies were carried out under different conditions: suspensions stored at 4 and 25 °C for 3 months, in SNF at 35 °C and in aCSF at 37 °C; dry powders were instead stored at 25 °C with 50 % relative humidity for 8 months. All the results demonstrated a greater stability of NPs in powder form and the absence of aggregation phenomena under artificial physiological conditions.

PALS1 is a key regulator of the lateral distribution of tight junction proteins in renal epithelial cells.

The evolutionarily conserved apical Crumbs (CRB) complex, consisting of the core components CRB3a (an isoform of CRB3), PALS1 and PATJ, plays a key role in epithelial cell-cell contact formation and cell polarization. Recently, we observed that deletion of one Pals1 allele in mice results in functional haploinsufficiency characterized by renal cysts. Here, to address the role of PALS1 at the cellular level, we generated CRISPR/Cas9-mediated PALS1-knockout MDCKII cell lines. The loss of PALS1 resulted in increased paracellular permeability, indicating an epithelial barrier defect. This defect was associated with a redistribution of several tight junction-associated proteins from bicellular to tricellular contacts. PALS1-dependent localization of tight junction proteins at bicellular junctions required its interaction with PATJ. Importantly, reestablishment of the tight junction belt upon transient F-actin depolymerization or upon Ca2+ removal was strongly delayed in PALS1-deficient cells. Additionally, the cytoskeleton regulator RhoA was redistributed from junctions into the cytosol under PALS1 knockout. Together, our data uncover a critical role of PALS1 in the coupling of tight junction proteins to the F-actin cytoskeleton, which ensures their correct distribution along bicellular junctions and the formation of tight epithelial barrier.

Successful Prediction of Fetal Exposure to Dual BCRP/P-gp Drug Substrates Using the Efflux Ratio-Relative Expression Factor Approach and PBPK M&S.

To inform fetal drug safety, it is important to determine or predict fetal drug exposure throughout pregnancy. The former is not possible in the first or second trimester. In contrast, at the time of birth, fetal drug exposure, relative to maternal exposure, can be estimated as Kp,uu (unbound fetal umbilical venous (UV) plasma area under the curve (AUC)/unbound maternal plasma (MP) AUC), provided the observed UV/MP values, spanning the dosing interval, are available from multiple maternal-fetal dyads. However, this fetal Kp,uu cannot be extrapolated to other drugs. To overcome the above limitations, we have used an efflux ratio-relative expression factor (ER-REF) approach to successfully predict the fetal Kp,uu of P-gp substrates. Because many drugs taken by pregnant people are also BCRP substrates, here, we extend this approach to drugs that are effluxed by both placental BCRP and P-gp or P-gp alone. To verify our predictions, we chose drugs for which UV/MP data were available at term: glyburide and imatinib (both BCRP and P-gp substrates) and nelfinavir (only P-gp substrate). First, the ER of the drugs was determined using Transwells and MDCKII cells expressing either BCRP or P-gp. Then, the ER was scaled using the proteomics-informed REF value to predict the fetal Kp,uu of the drug at term. The ER-REF predicted fetal Kp,uu of glyburide (0.43), imatinib (0.42), and nelfinavir (0.40) fell within two-fold of the corresponding in vivo fetal Kp,uu (0.44, 0.37, and 0.46, respectively). These data confirm that the ER-REF approach can successfully predict fetal drug exposure to BCRP/P-gp and P-gp substrates, at term.

Abstract A139: Preclinical characterization and prediction of human pharmacokinetics and efficacious dose for VIP236, a novel alpha V beta 3 binding small molecule-drug conjugate (SMDC)

Abstract VIP236 is a small molecule drug conjugate (SMDC) consisting of an alpha v beta 3 (αvβ3) integrin binder linked to an optimized camptothecin (optCPT) payload via a protease sensitive peptide sequence. The overall drug design strategy for VIP236 is targeted delivery to the tumor microenvironment (TME) with extracellular release of the opCPT by neutrophil elastase (NE) present in the TME. VIP236 is anticipated to kill tumor cells while reducing effects to normal tissue. VIP236 was characterized in preclinical pharmacokinetic (PK) and efficacy studies to predict its human PK and efficacious dose. Briefly, VIP236 was administered intravenously (IV) to mice, rats, and dogs to characterize both its in vivo PK (1-2 mg/kg) and its routes of excretion (bile-duct cannulated [BDC] rat at 10 mg/kg). VIP236 was evaluated in vitro for its protein binding characteristics and as a substrate of human drug transporters. In addition, VIP236 was dosed to MX-1 (breast cancer) xenografted mice at doses ranging from 26-40 mg/kg IV on a 3 days on/4 days off regimen to characterize its antitumor activity. VIP236 had low plasma clearance in mice, rats, and dogs (CL=2.7-4.4 mL/min/kg) and low volume of distribution (Vss = 0.11 to 0.25 L/kg). Terminal half-life ranged from 0.93 hour in mice to 3.07 hours in dogs. In BDC rats, VIP236 was primarily excreted in bile with biliary clearance accounting for essentially all the total body CL. There was minimal to no metabolites in rat bile and urine. In vitro VIP236 exhibited low permeability in MDCKII cell monolayers and was identified as a substrate of BSEP and MRP2. VIP236 was highly protein bound to plasma protein with fraction unbound being <5% in all species tested. VIP236 showed robust tumor regression in MX-1 xenografted mice at all doses tested. Allometric methods were used to predict a low human VIP236 CL (0.87-2.68 mL/min/kg) and Vss (0.10 L/kg). Translational pharmacokinetic/pharmacodynamic modeling was used to predict human efficacious doses and exposures by benchmarking VIP236 against irinotecan antitumor activity in MX-1 xenograft mice after 5 cycles of dosing. Based on this analysis, predicted human doses ranged from 1-5 mg/kg IV on a 2 days on/5 days off regimen. VIP236 is currently being evaluated in a first-in-human study in patients with advanced or metastatic solid tumors (NCT05712889). Citation Format: Harvey Wong, Beatrix Stelte-Ludwig, Hans-Georg Lerchen, Amy J Johnson, Melanie M Frigault, Raquel Izumi, Ahmed Hamdy. Preclinical characterization and prediction of human pharmacokinetics and efficacious dose for VIP236, a novel alpha V beta 3 binding small molecule-drug conjugate (SMDC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A139.

Investigation into the impact of proton pump inhibitors on sertraline transport across the blood-brain barrier

As a widely used antidepressant that works by inhibiting the reuptake of serotonin, sertraline exerts an antidepressant effect depending on its concentration in the brain, which might be limited by the blood-brain barrier (BBB). It is highly possible to combine proton pump inhibitors (PPIs) with sertraline in clinical trials. Nevertheless, the role played by PPIs in regulating the transport of sertraline across the BBB remains unclear. Here, the impact of PPIs on the distribution of sertraline in the brain and the mechanisms involved were investigated. A mouse brain distribution study showed that Omeprazole (OME), Pantoprazole (PAN), Ilaprazole (ILA), and Esomeprazole (ESO) increased the area under the brain concentration-time curves (AUC) for sertraline by 2.02-, 3.18-, 3.04-, and 4.21-fold, respectively, after the 14-day administration of PPIs. Besides, PPIs significantly increased the permeability of sertraline in brain perfusion experiments, with PAN having the highest rank order, followed by ILA, OME, and ESO. In the tail suspension test (TST), co-administration PPI groups showed significantly shorter immobility time than the control group. In vitro, four PPIs inhibited sertraline efflux in breast cancer resistance protein (BCRP)-overexpressing MDCKII cells, and showed a mixed inhibition type. In this study, PPIs were further found to inhibit the mRNA and protein expression of brain BCRP. To sum up, the findings of this study revealed that PPIs could enhance the brain distribution and antidepressant effect of sertraline, which may be attributed to the inhibition of BCRP expression at the BBB by PPIs.

Potentiation of the Uricosuric Effect of Dotinurad by Trans-Inhibition of the Uric Acid Reabsorptive Transporter URAT1.

URAT1 is a transporter responsible for uric acid (UA) reabsorption by renal proximal tubules and a pharmacological target of uricosuric agents. Probenecid and benzbromarone have been used as uricosuric agents, while dotinurad was recently approved in Japan. Notably, the in vitro inhibition potential (IC 50) of dotinurad on URAT1 is not strong enough to explain its in vivo uricosuric effect estimated based on clinical unbound plasma concentrations, suggesting the presence of mechanisms other than competition with UA uptake at the extracellular domain of URAT1 (cis-inhibition). In this study, trans-inhibition was hypothesized as the mechanism underlying URAT1 inhibition by dotinurad, wherein intracellularly accumulated dotinurad inactivates URAT1. In URAT1-expressing MDCK-II cells and Xenopus oocytes, pre-incubation with dotinurad potentiated the inhibitory effect more than co-incubation alone, but this effect was not observed with benzbromarone or probenecid. Under co-incubation, dotinurad inhibited UA uptake in a competitive manner (cis-inhibition). When we pre-injected dotinurad directly into oocytes and immediately measured [14C]UA uptake without co-incubation (only trans-inhibition), dotinurad noncompetitively inhibited UA uptake. URAT1 is an exchange transporter for UA and monocarboxylates such as nicotinic acid (NA). Pre-injected dotinurad and extracellular UA attenuated and facilitated efflux of [3H]NA, respectively, whereas pre-injection of benzbromarone or probenecid did not affect it, suggesting that dotinurad exhibits trans-inhibition by attenuating URAT1-mediated efflux of monocarboxylates, which is a driving force for UA uptake by URAT1. Accordingly, dotinurad ameliorates URAT1-mediated UA reabsorption by both cis- and trans-inhibition, explaining its clinically stronger uricosuric effect than that estimated by the in vitro IC 50 value. Significance Statement The uricosuric agent dotinurad inhibits uric acid reabsorptive transporter URAT1 with a clinical potency stronger than that estimated from IC 50 obtained by in vitro URAT1 inhibition. This in vivo-in vitro discrepancy was explained by the trans-inhibition effect of dotinurad on URAT1. Trans-inhibition was due to the attenuation of monocarboxylates efflux via URAT1, which is a driving force for URAT1-mediated exchange transport of uric acid. Overall, this is the first study to experimentally demonstrate trans-inhibition mechanism of URAT1.

Microbial metabolite n-butyrate upregulates intestinal claudin-23 expression through SP1 and AMPK pathways in mouse colon and human intestinal Caco-2 cells

AimsRegulation of the intestinal barrier is closely related to intestinal microbial metabolism. This study investigated the role of intestinal microflora in the regulation of the tight junction (TJ) barrier in epithelial cells, focusing on the microbial metabolite n-butyrate, a major short-chain fatty acid, using mice and human intestinal Caco-2 cells. Materials and methodsWhole transcriptome analysis with RNA sequencing and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were performed in the colon of germ-free (GF) and specific pathogen-free (SPF) mice. Claudin-23 expression was examined by qRT-PCR, immunoblotting, and immunofluorescence in Caco-2 cells treated with n-butyrate. Luciferase reporter assay was performed to examine the effect of n-butyrate on claudin-23 transcriptional activity. The siRNA targeting the transcription factor SP1 and pharmacological inhibitor of AMPK were used in combination. TJ permeability was examined in canine kidney MDCKII cells stably expressing human claudin-23. Key findingsCldn23 mRNA expression was downregulated in the colon of GF mice (0.6-fold) compared to that in SPF mice. n-Butyrate upregulated claudin-23 mRNA (1.7-fold) and protein (2.1-fold) expression as well as increased the transcriptional activity (15-fold) of CLDN23 in Caco-2 cells. The n-butyrate-mediated increase in claudin-23 expression and transcriptional activity was reduced by inhibition of SP1 and AMPK. Exogenously expressed human claudin-23 in MDCKII cells did not affect TJ permeability to ions and macromolecules. Significancen-Butyrate regulates intestinal claudin-23 expression through the SP1 and AMPK pathways. This mechanism may be involved in the beneficial effects of n-butyrate-mediated intestinal homeostasis.

Abstract 5060: Tucatinib does not alter oxaliplatin PK or associated renal function: An OCT2 and MATE transport inhibition study

Abstract Background: Oxaliplatin (OX)-containing regimens are frequently utilized to treat gastrointestinal (GI) cancers. OX is eliminated predominately via urinary excretion (GFR and active tubular secretion). The contribution of active transport via OCT2 and MATE1/2-K to OX clearance is not fully understood. Tyrosine kinase inhibitors (TKIs) are frequently found to interact with OCT2/MATE transporters; however, a gap in knowledge remains between their clinical potential to impact OX pharmacokinetics (PK) and in turn, impact renal function. Tucatinib (TUC) is a highly selective human epidermal growth factor receptor 2 (HER2)-directed TKI approved in multiple regions in combination with trastuzumab and capecitabine for adult patients with metastatic HER2+ breast cancer and is currently being investigated in other HER2+ tumors. TUC inhibits OCT2/MATE-mediated transport of metformin and creatinine in vitro and in vivo. In this study, we investigated the impact of TUC on OX plasma PK, OX renal clearance (Clr), and renal function. Methods: In vitro inhibition of OCT2/MATE-mediated transport of OX by TUC was assessed in OCT2, MATE1, or MATE2-K-expressing MDCK-II cells. SGNTUC-024 (NCT04430738) is a Ph1b/2 clinical study in patients with HER2+ GI cancers evaluating the impact of TUC on the safety and PK of OX. Patients received TUC 150 mg (Cohort 1A) or 300 mg (Cohort 1B) BID starting on C1D8 of a 2-week cycle in combination with modified FOLFOX6/7. Intensive PK was collected in plasma and urine for OX alone (C1D1) or with steady-state TUC (C2D1). Total plasma platinum (Pt, analyzed as a surrogate for OX and catabolites), plasma Pt ultrafiltrate (PUF, unbound), and urine Pt were quantitatively analyzed via ICP-MS. Serum Cystatin C (CysC) was measured as a pharmacodynamic (PD) renal function marker in Cohorts 1A and 1B. Results: TUC inhibited in vitro OX transport by MATE1 (IC50 = 0.0639 µM), MATE2-K (IC50 = 0.0382 µM), and OCT2 (IC50 = 0.491 µM). In 11 patients, total Pt and PUF AUC0-8h geometric mean ratio (GMR) and 90% confidence intervals (CI) between patients who received OX alone compared to in combination were 1.1 (0.98, 1.3) and 1.0 (0.78, 1.4) in Cohort 1A (n=4) and 1.1 (0.96, 1.2) and 1.0 (0.98, 1.1) in Cohort 1B (n=7), respectively. OX GM (%CV) renal clearance (Clr,0-8h in mL/min) and fraction excreted 24h post-dose (fe,0-24h) were similar with and without TUC in both Cohort 1A (C1D1 Clr0-8h = 277 (33), fe,0-24h = 22% (17); C2D1 Clr0-8h = 249 (78), fe,0-24h = 21% (15)) and Cohort 1B (C1D1 Clr0-8h = 189 (37), fe,0-24h = 17.7% (16); C2D1 Clr0-8h = 177 (40), fe,0-24h = 16.6% (35)). Reversible slight increases in CysC (normalized to baseline) on day 3 of each cycle, irrespective of tucatinib, were observed. Conclusions: This investigation of in vitro and in vivo determinants of OX PK demonstrates that TUC does not alter the renal clearance of oxaliplatin nor renal function when OX is administered in combination with TUC. Citation Format: Ariel R. Topletz-Erickson, Anthony Lee, Vineet Kumar, Michelle Ubowski, JoAl G. Mayor, Layth I. Abdulrasool, Clark M. Henderson, Joseph A. Ware, Christopher J. Endres. Tucatinib does not alter oxaliplatin PK or associated renal function: An OCT2 and MATE transport inhibition study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5060.

Cellular segregation in cocultures is driven by differential adhesion and contractility on distinct timescales

Cellular sorting and pattern formation are crucial for many biological processes such as development, tissue regeneration, and cancer progression. Prominent physical driving forces for cellular sorting are differential adhesion and contractility. Here, we studied the segregation of epithelial cocultures containing highly contractile, ZO1/2-depleted MDCKII cells (dKD) and their wild-type (WT) counterparts using multiple quantitative, high-throughput methods to monitor their dynamical and mechanical properties. We observe a time-dependent segregation process governed mainly by differential contractility on short (<5 h) and differential adhesion on long (>5 h) timescales. The overly contractile dKD cells exert strong lateral forces on their WT neighbors, thereby apically depleting their surface area. Concomitantly, the tight junction-depleted, contractile cells exhibit weaker cell-cell adhesion and lower traction force. Drug-induced contractility reduction and partial calcium depletion delay the initial segregation but cease to change the final demixed state, rendering differential adhesion the dominant segregation force at longer timescales. This well-controlled model system shows how cell sorting is accomplished through a complex interplay between differential adhesion and contractility and can be explained largely by generic physical driving forces.

In vitro interaction of the pesticides flupyradifurone, bupirimate and its metabolite ethirimol with the ATP-binding cassette transporter G2 (ABCG2)

ABCG2 is an ATP-binding cassette efflux transporter that is expressed in absorptive and excretory organs such as liver, intestine, kidney, brain and testis where it plays a crucial physiological and toxicological role in protecting cells against xenobiotics, affecting pharmacokinetics of its substrates. In addition, the induction of ABCG2 expression in mammary gland during lactation is related to active secretion of many toxicants into milk. In this study, the in vitro interactions between ABCG2 and three pesticides flupyradifurone, bupirimate and its metabolite ethirimol were investigated to check whether these compounds are substrates and/or inhibitors of this transporter. Using in vitro transepithelial assays with cells transduced with murine, ovine and human ABCG2, we showed that ethirimol and flupyradifurone were transported efficiently by murine Abcg2 and ovine ABCG2 but not by human ABCG2. Bupirimate was not found to be an in vitro substrate of ABCG2 transporter. Accumulation assays using mitoxantrone in transduced MDCK-II cells suggest that none of the tested pesticides were efficient ABCG2 inhibitors, at least in our experimental conditions. Our studies disclose that ethirimol and flupyradifurone are in vitro substrates of murine and ovine ABCG2, opening the possibility of a potential relevance of ABCG2 in the toxicokinetics of these pesticides.

Improvement of Workflows and Assay Reproducibility by The Introduction of "Assay-Ready" Culturing of MDCK Cells for Transport Studies.

In previous studies, we established and validated three Madin Darby Canine Kidney MDCKII cell lines, recombinantly modified with zinc finger nuclease (ZFN) technology. Here, we investigated the applicability of seeding these three canine P-gp deficient MDCK_ZFN cell lines, directly from frozen cryopreserved stocks without previous cultivation for efflux transporter and permeability studies. This technique is referred to as "assay-ready" and allows for highly standardized conduction of cell-based assays and shorter cultivation cycles. To obtain a rapid fitness of the cells for that purpose, a very gentle freezing and thawing protocol was applied. Assay-ready MDCK_ZFN cells were tested in bi-directional transport studies and compared to their traditionally cultured counterparts. Long-term performance robustness, human effective intestinal permeability (Peff) predictability and batch to batch variability were assessed. Efflux ratios (ER) and apparent permeability (Papp) results were highly comparable between assay-ready and standard cultured cell lines with R2 values of 0.96 or higher. Papp to Peff correlations obtained from passive permeability with non-transfected cells were comparable independent of the cultivation regime. Long-term evaluation revealed robust performance of assay-ready cells and reduced data variability of reference compounds in 75% of cases compared to standard cultured MDCK_ZFN cells. Assay-ready methodology for handling MDCK_ZFN cells allows more flexibility in assay planning and reduces performance fluctuations in assays caused by cell aging. Therefore, the assay-ready principle has proven superior over conventional cultivation for MDCK_ZFN cells and is considered as a key technology to optimize processes with other cellular systems.

Modulatory Activity of the Copper Chelate, Copper N-(2-Hydroxy Acetophenone) Glycinate, in ABC-transporter-expressing Cell Lines.

Metal-containing compounds (e.g., platinum complexes) belong to the standard armamentarium of cancer chemotherapy. Copper N-(2-hydroxy acetophenone) glycinate (CuNG) exerts anticancer activity in vitro and in vivo and modulates drug resistance related to glutathione or P-glycoprotein. The potential of CuNG to interact with ATP-binding cassette (ABC) transporters has not been fully explored yet. This study focused on the modulatory effects of CuNG on four ABC transporters (MRP1, MRP1, BCRP, and P-glycoprotein). Cell viability, drug uptake and ABC transporter expression were measured by resazurin assays, flow cytometry, and ELISA in HL60AR, MDCKII-hBCRP, and Caco-2 cells. CuNG increased doxorubicin sensitivity of MRP1-over-expressing HL60AR with a similar efficacy as the control MRP1 inhibitor MK571. CuNG also increased MRP1's efflux activity. Comparable results were obtained with MDCKII cells over-expressing hBCRP. ELISA assays revealed that the expression of MRP1 in HL60AR cells and BCRP in MDCKII- cells was predominant but other ABC-transporters were also expressed at lower levels. Caco-2 cells expressed high levels of MRP2, but MRP1, BCRP, and P-glycoprotein were also expressed. In contrast to the two former cell lines, CuNG increased doxorubicin resistance and decreased efflux activity in Caco-2 cells. CuNG exerted different modulatory activities towards ABC-transporter-expressing cells. While CuNG-mediated ABC-transporter inhibition may improve tumor chemotherapy (like in HL60AR and MDCKII-hBCRP cells), CuNG-mediated enhanced ABC-transport (like in Caco-2 cells) may be a new strategy to ameliorate inflammatory diseases associated with decreased ABC-transporter expression such as ulcerative colitis.

Propionate regulates tight junction barrier by increasing endothelial-cell selective adhesion molecule in human intestinal Caco-2 cells

Regulation of the intestinal barrier is closely associated with intestinal microbial metabolism. This study investigated the role of propionate, a major short-chain fatty acid produced by intestinal microorganisms, in the regulation of the tight junction (TJ) barrier in human intestinal Caco-2 cells. Propionate strengthened TJ barrier integrity, as indicated by decreased permeability to macromolecules and increased transepithelial electrical resistance in Caco-2 cells. DNA microarray analysis revealed that propionate upregulated endothelial cell-selective adhesion molecule (ESAM), a TJ-associated protein, without any increase in other TJ proteins. The upregulation of ESAM was confirmed using quantitative reverse transcription-PCR, immunoblotting, and immunofluorescence analyses. Luciferase promoter analysis demonstrated that propionate induced the transcriptional activation of ESAM. The effects of propionate were sensitive to nilotinib inhibition of NR2C2. Overexpression of human ESAM (hESAM) in canine kidney epithelial MDCK-II cells lowered the permeability to macromolecules in a manner similar to that of propionate-treated Caco-2 cells. hESAM overexpression facilitated calcium-induced assembly of the TJ complex in MDCK-II cells. Taken together, propionate strengthened the intestinal TJ barrier by increasing ESAM levels in Caco-2 cells.

A Unique In Vitro Assay to Investigate ABCB4 Transport Function

ABCB4 is almost exclusively expressed in the liver, where it plays an essential role in bile formation by transporting phospholipids into the bile. ABCB4 polymorphisms and deficiencies in humans are associated with a wide spectrum of hepatobiliary disorders, attesting to its crucial physiological function. Inhibition of ABCB4 by drugs may lead to cholestasis and drug-induced liver injury (DILI), although compared with other drug transporters, there are only a few identified substrates and inhibitors of ABCB4. Since ABCB4 shares up to 76% identity and 86% similarity in the amino acid sequence with ABCB1, also known to have common drug substrates and inhibitors, we aimed to develop an ABCB4 expressing Abcb1-knockout MDCKII cell line for transcellular transport assays. This in vitro system allows the screening of ABCB4-specific drug substrates and inhibitors independently of ABCB1 activity. Abcb1KO-MDCKII-ABCB4 cells constitute a reproducible, conclusive, and easy to use assay to study drug interactions with digoxin as a substrate. Screening a set of drugs with different DILI outcomes proved that this assay is applicable to test ABCB4 inhibitory potency. Our results are consistent with prior findings concerning hepatotoxicity causality and provide new insights for identifying drugs as potential ABCB4 inhibitors and substrates.

Assessment of the role of nucleoside transporters, P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated protein 2 in the placental transport of entecavir using in vitro, ex vivo, and in situ methods

The nucleoside analog entecavir (ETV) is a first-line pharmacotherapy for chronic hepatitis B in adult and pediatric patients. However, due to insufficient data on placental transfer and its effects on pregnancy, ETV administration is not recommended for women after conception. To expand knowledge of safety, we focused on evaluating the contribution of nucleoside transporters (NBMPR sensitive ENTs and Na+ dependent CNTs) and efflux transporters, P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), and multidrug resistance-associated transporter 2 (ABCC2), to the placental kinetics of ETV. We observed that NBMPR and nucleosides (adenosine and/or uridine) inhibited [3H]ETV uptake into BeWo cells, microvillous membrane vesicles, and fresh villous fragments prepared from the human term placenta, while Na+ depletion had no effect. Using a dual perfusion study in an open-circuit setup, we showed that maternal-to-fetal and fetal-to-maternal clearances of [3H]ETV in the rat term placenta were decreased by NBMPR and uridine. Net efflux ratios calculated for bidirectional transport studies performed in MDCKII cells expressing human ABCB1, ABCG2, or ABCC2 were close to the value of one. Consistently, no significant decrease in fetal perfusate was observed in the closed-circuit setup of dual perfusion studies, suggesting that active efflux does not significantly reduce maternal-to-fetal transport. In conclusion, ENTs (most likely ENT1), but not CNTs, ABCB1, ABCG2, and ABCC2, contribute significantly to the placental kinetics of ETV. Future studies should investigate the placental/fetal toxicity of ETV, the impact of drug-drug interactions on ENT1, and interindividual variability in ENT1 expression on the placental uptake and fetal exposure to ETV.

Transfer-Tattoo-Like Cell-Sheet Delivery Induced by Interfacial Cell Migration.

A direct transfer of a cell sheet from a culture surface to a target tissue is introduced. Commercially available, flexible parylene is used as the culture surface, and it is proposed that the UV-treated parylene offers adequate and intermediate levels of cell adhesiveness for both the stable cell attachment during culture and for the efficient cell transfer to a target surface. The versatility of this cell-transfer process is demonstrated with various cell types, including MRC-5, HDFn, HULEC-5a, MC3T3-E1, A549, C2C12 cells, and MDCK-II cells. The novel cell-sheet engineering is based on a mechanism of interfacial cell migration between two surfaces with different adhesion preferences. Monitoring of cytoskeletal dynamics and drug treatments during the cell-transfer process reveals that the interfacial cell migration occurs by utilizing the existing transmembrane proteins on the cell surface to bind to the targeted surface. The re-establishment and reversal of cell polarity after the transfer process are also identified. Its unique capabilities of 3D multilayer stacking, freeform design, and curved surface application are demonstrated. Finally, the therapeutic potential of the cell-sheet delivery system is demonstrated by applying it to cutaneous wound healing and skin-tissue regeneration in mice models.