MDCKII Cells Research Articles

P03.09 * PHARMACOLOGICAL MODULATION OF BLOOD-BRAIN BARRIER: FUTURE STRATEGY FOR TREATMENT OF BRAIN TUMORS

A prerequisite for the efficacy of chemotherapy is that it reaches the tumor mass at a therapeutic concentration. In brain tumors this phenomenon is hampered by the presence of the blood brain barrier (BBB) which limits the spread of chemotherapeutic agents within the brain. It is lately emerged as this Multi Drug Resistance (MDR) phenomenon is explained through the cooperation of P-glycoprotein (P-gp, ABCB1) and breast cancer resistance protein (BCRP, ABCG2), two “gatekeeper transporters that work in tandem on the BBB and are also present on the plasma membrane of certain brain tumors. Recently, we have attempted to improve the therapeutic efficacy of pharmacological treatment in malignant brain tumors by safe and temporary BBB permeabilization. We have demonstrated that morphine and, to a lower level, ondansetron and dexamethasone allow an accumulation of doxorubicin within the rat brain by LC-MS/MS mass spectrometry. All these drugs are substrates of P-gp and BCRP efflux pumps. The aim of the current proposal is to expand our preliminary observation, to understand the mechanism of action of BBB “permeabilization” induced by morphine or other drugs, and to exploit this method for the “treatment” of brain tumour in an animal model. i) Quantifying the level drugs that do not usually cross the BBB (mitoxantrone or melphalan) after morphine pre-treatment in a preclinical model. Verifying the cytotoxic effect of morphine plus doxorubicin (and other chemotherapeutic agents) treatment by using MTT and TUNEL analysis in glioblastoma cell lines ii) Quantifying the level of drugs that do not usually cross the BBB after morphine pre-treatment in an artificial BBB through a monolayer of MDCKII cells over-expressing the human P-gp or BCRP. iii) Investigating the regulatory role of certain microRNA in MDR mechanism by RT-PCR and western blot analysis of P-gp, BCRP, miR-21, miR-27a and miR-451. Our data suggest that blocking efflux transporters by pretreatment with morphine, ondansetron or desamethasone is able to allow doxorubicin penetration inside the brain. This is not associated with acute cardiac or renal toxicity. These preliminary results will enable us to novel therapeutic approaches to refractory or recurrent brain tumors in which molecules usually stopped by the BBB may have a therapeutic impact.

Hop-derived prenylflavonoids are substrates and inhibitors of the efflux transporter breast cancer resistance protein (BCRP/ABCG2).

Hops (Humulus lupulus L.) produce unique prenylflavonoids that exhibit interesting bioactivities. This study investigates the interactions between selected prenylflavonoids and breast cancer resistance protein (BCRP/ABCG2), an efflux transporter important for xenobiotic bioavailability and multidrug resistance (MDR). ABCG2-inhibitory activity of xanthohumol (XN), isoxanthohumol (IX), 6-prenylnaringenin (6-PN), 8-prenylnaringenin (8-PN), and 6,8-diprenylnarigenin (6,8-diPN) was evaluated using mitoxantrone accumulation and vesicular transport assays. XN, IX, and 8-PN were tested for a substrate-type relationship with ABCG2 using ATPase and bidirectional transport assays. The prenylflavonoids exhibited significant ABCG2-inhibitory activities in mitoxantrone accumulation and vesicular transport assays. In the ATPase assay, XN, IX, and 8-PN inhibited baseline and sulfasalazine-stimulated ATPase activities with IC50 of 2.16-27.0μM. IX and 8-PNalso displayed bell-shaped activation curves in Ko143-suppressed membranes, indicating a substrate-type relationship. For IX, efflux ratios of 1.25 ± 0.21 and 9.18 ± 0.56 were observed in wild type and ABCG2-overexpressing MDCKII cell monolayers, respectively. The latter was reduced to 1.25 ± 0.15 in the presence of the ABCG2-specific inhibitor Ko143, demonstrating an ABCG2-mediated efflux of IX. Additionally, evidence was shown for the involvement of ABCG2 in the efflux of 8-PN and/or its sulfate conjugate. Prenylflavonoids are potent inhibitors of ABCG2 and therefore implicated in ABCG2-mediated food/herb-drug interactions and MDR. ABCG2-mediated efflux of prenylflavonoids may represent one mechanism that regulates prenylflavonoid bioavailability.

"Effect of the drug transporters ABCB1, ABCC2, and ABCG2 on the disposition and brain accumulation of the taxane analog BMS-275,183".

BMS-275,183 is a novel oral C-4 methyl carbonate analogue of paclitaxel. Recently, a drug-drug interaction between BMS-275,183 and benzimidazole proton pump inhibitors (PPIs) was suggested in clinical trials resulting in elevated drug exposure and toxicity. We explored whether the interaction takes place at the level of P-glycoprotein (Pgp, MDR1, ABCB1), Breast Cancer Resistance Protein (BCRP, ABCG2) and MRP2 (ABCC2) using in vitro and in vivo models. In vitro cell survival, drug accumulation, efflux and transport studies with BMS-275,183 were performed employing MDCKII (wild-type, MDR1, BCRP, MRP2) and LLCPK (wild-type and MDR1) cells. In vivo the pharmacokinetics and tissue distribution of BMS-275,183 after p.o. and i.v. administration were explored in Mdr1a/1b(-/-) and wild-type mice, in presence or absence of the PPI pantoprazole. Results In vitro, BMS-275,183 was found to be a good substrate for MDR1, a moderate substrate for MRP2 and not a substrate for BCRP. In vivo, oral bioavailability, plasma AUC0-6h and brain concentrations were significantly 1.5-, 4-, and 2-fold increased, respectively, in Mdr1a/1b(-/-) compared with wild-type mice (p < 0.001). However, oral co-administration of pantoprazole (40 mg/kg) did not alter the pharmacokinetics of BMS-275,183 in wild-type mice. Conclusions BMS-275,183 is efficiently transported by Pgp and to a lesser extent by MRP2 in vitro. Genetic deletion of Pgp significantly altered the pharmacokinetics and brain distribution of p.o. and i.v. administered BMS-275,183 in Mdr1a/1b-/- compared to wild-type mice. Oral co-administration of BMS-275,183 with pantoprazole did not affect the pharmacokinetics of BMS-275,183 in wild-type mice, suggesting no interaction with PPI at the dose employed.

The need for human breast cancer resistance protein substrate and inhibition evaluation in drug discovery and development: why, when, and how?

Although the multiplicity in transport proteins assessed during drug development is continuously increasing, the clinical relevance of the breast cancer resistance protein (BCRP) is still under debate. Here, our aim is to rationalize the need to consider BCRP substrate and inhibitor interactions and to define optimum selection and acceptance criteria between cell-based and vesicle-based assays in vitro. Information on the preclinical and clinical pharmacokinetics (PK), drug-drug interactions, and pharmacogenomics data was collated for 13 marketed drugs whose PK is reportedly associated with BCRP interaction. Clinical examples where BCRP impacts drug PK and efficacy appear to be rare and confounded by interactions with other transporters. Thirty-seven compounds were selected to be tested as BCRP substrates in a cell-based assay using MDCKII cells (Madin-Darby canine kidney cells) and 18 in membrane vesicles. Depending on the physicochemical compound properties, we observed both in vitro systems to give false-negative readouts. In addition, the inhibition potential of 19 compounds against BCRP was assessed in vesicles and in MDCKII cells, where we observed significant system and substrate-dependent IC50 values. Therefore, neither of the two test systems is superior to the other. Instead, one system may offer advantages under certain situations (e.g., low permeability) and thus should be selected based on the physicochemical compound properties. Finally, given the clinical relevance of BCRP, we propose that its evaluation should remain issue-driven: for low permeable, low bioavailable drugs, in particular when other more common processes do not allow a mechanistic understanding of any unexpected absorption or brain disposition, and for drugs with a low therapeutic window.

Analysis of Pseudoxanthoma Elasticum–Causing Missense Mutants of ABCC6 In Vivo; Pharmacological Correction of the Mislocalized Proteins

Mutations in the ABCC6 gene cause soft tissue calcification in pseudoxanthoma elasticum (PXE) and in some patients generalized arterial calcification of infancy (GACI). PXE is characterized by late-onset and progressive mineralization of elastic fibers in dermal, ocular and cardiovascular tissues. GACI patients present a more severe, often prenatal arterial calcification. We have tested ten frequent disease-causing ABCC6 missense mutants for the transport activity using Sf9 cells, characterized the subcellular localization in MDCKII cells and in mouse liver, and tested the phenotypic rescue in zebrafish. We aimed at identifying mutants with preserved transport activity but with improper plasma membrane localization for rescue by the chemical chaperone 4-phenylbutyrate (4-PBA). Seven of the mutants were transport-competent but mislocalized in mouse liver. The observed divergence in cellular localization of mutants in MDCKII cells vs. mouse liver underlined the limitations of this two-dimensional in vitro cell system. The functionality of ABCC6 mutants was tested in zebrafish and minimal rescue of the morpholino-induced phenotype was found. However, 4-PBA, a drug approved for clinical use, restored the plasma membrane localization of four ABCC6 mutants (R1114P, S1121W, Q1347H, R1314W), suggesting that allele-specific therapy may be useful for selected patients with PXE and GACI.

In vitro transport assays of rufinamide, pregabalin, and zonisamide by human P-glycoprotein

Epilepsy is resistant to treatment with antiepileptic drugs (AEDs) in about one third of epilepsy patients. AED export by P-glycoprotein (Pgp) overexpressed in the blood-brain barrier may contribute to AED resistance. The Pgp transport status of many of the recently approved AEDs remains unknown. We investigated whether several new AEDs - zonisamide (ZNS), pregabalin (PGB), and rufinamide (RFM) - are human Pgp substrates. MDCKII and LLC-PK1 cells transfected with the human MDR1 gene, which encodes the Pgp protein, were used in concentration equilibrium transport assays (CETA) to determine the substrate status of ZNS, PGB, and RFM. For each drug, an equal concentration was added to apical and basal chambers, and the concentration in both chambers was measured up to 4h. RFM, ZNS, and PGB were not transported by MDR1-transfected cells from basolateral to apical sides in CETA. ZNS, RFM, and PGB are not substrates of human Pgp. These data suggest that resistance to these drugs may not be attributed to increased Pgp activity in resistant patients.

Interaction of Novel Platelet-Increasing Agent Eltrombopag with Rosuvastatin via Breast Cancer Resistance Protein in Humans

Eltrombopag (ELT), an orally available thrombopoietin receptor agonist, is a substrate of organic anion transporting polypeptide 1B1 (OATP1B1), and coadministration of ELT increases the plasma concentration of rosuvastatin in humans. Since the pharmacokinetic mechanism(s) of the interaction is unknown, the present study aimed to clarify the drug interaction potential of ELT at transporters. The OATP1B1-mediated uptake of ELT was inhibited by several therapeutic agents used to treat lifestyle diseases. Among them, rosuvastatin was a potent inhibitor with an IC(50) of 0.05 µM, which corresponds to one-seventh of the calculated maximum unbound rosuvastatin concentration at the inlet to the liver. Nevertheless, a simulation study using a physiologically based pharmacokinetic model predicted that the effect of rosuvastatin on the pharmacokinetic profile of ELT in vivo would be minimal. On the other hand, ELT potently inhibited uptake of rosuvastatin by OATP1B1 and human hepatocytes, with an IC(50) of 0.1 µM. However, the results of the simulation study indicated that inhibition of OATP1B1 by ELT can only partially explain the clinically observed interaction with rosuvastatin. ELT also inhibited transcellular transport of rosuvastatin in MDCKII cells stably expressing breast cancer resistance protein (BCRP), and was found to be a substrate of BCRP. The interaction of ELT with rosuvastatin can be almost quantitatively explained on the assumption that intestinal secretion of rosuvastatin is essentially completely inhibited by ELT. These results suggest that BCRP in small intestine may be the major target for interaction between ELT and rosuvastatin in humans.

OATP1B3 Is Expressed in Pancreatic β-Islet Cells and Enhances the Insulinotropic Effect of the Sulfonylurea Derivative Glibenclamide

Organic anion transporting polypeptide OATP1B3 is a membrane-bound drug transporter that facilitates cellular entry of a variety of substrates. Most of the previous studies focused on its hepatic expression and function in hepatic drug elimination. In this study, we report expression of OATP1B3 in human pancreatic tissue, with the abundance of the transporter localized in the islets of Langerhans. Transport studies using OATP1B3-overexpressing MDCKII cells revealed significant inhibition of the cellular uptake of the known substrate cholecystokinin-8 in the presence of the insulinotropic antidiabetes compounds tolbutamide, glibenclamide, glimepiride, and nateglinide and identified glibenclamide as a novel substrate of OATP1B3. Sulfonylurea derivatives exert their insulinotropic effect by binding to the SUR1 subunit of the KATP channels inducing insulin secretion in β-cells. Here, we show that transient overexpression of human OATP1B3 in a murine β-cell line (MIN6)-which exhibits glucose and glibenclamide-sensitive insulin secretion-significantly enhances the insulinotropic effect of glibenclamide without affecting glucose-stimulated insulin secretion. Taken together, our data provide evidence that the drug transporter OATP1B3 functions as a determinant of the insulinotropic effect of glibenclamide on the tissue level. Changes in transport activity based on drug-drug interactions or genetic variability may therefore influence glibenclamide efficacy.

A novel MDCKII in vitro model for assessing ABCG2-drug interactions and regulation of ABCG2 transport activity in the caprine mammary gland by environmental pollutants and pesticides

The ABC efflux transporter ABCG2 represents the main route for active secretion of xenobiotics into milk. Thus, ABCG2 regulation by aryl hydrocarbon receptor (AhR) ligands including ubiquitously environmental pollutants is of great toxicological relevance. However, no adequate in vitro model is as yet available to study AhR-dependent ABCG2 regulation in dairy animals. In this study, we therefore systematically investigated the effect of various environmental contaminants and pesticides on ABCG2 efflux activity in MDCKII cells stably expressing mammary ABCG2 from dairy goats. The AhR-agonists TCDD, Aroclor 1254, prochloraz, and iprodione caused a dose- and time-dependent increase in EROD activity. Moreover, TCDD and prochloraz significantly stimulated ABCG2 transport activity through a dose- and time-dependent induction of transporter gene expression. AhR inhibitors like CH223191 significantly reversed TCDD- and prochloraz-induced stimulation of ABCG2 efflux activity. In contrast, non-AhR activators such as PCB 101 had no significant effect on EROD activity, ABCG2 gene expression or transporter activity. As we identified various anthelmintics including monepantel as potential ABCG2 substrates this regulatory mechanism may result in increased milk residues of potentially harmful xenobiotics. Thus, MDCKII-cABCG2 cells may represent a suitable in vitro model to study mammary ABCG2 secretory activity and its potential regulation by AhR-activating contaminants.

Interactions of tenofovir and tenofovir disoproxil fumarate with drug efflux transporters ABCB1, ABCG2, and ABCC2; role in transport across the placenta

Tenofovir (TFV) is used in pregnant women as a part of combination antiretroviral treatment to prevent mother-to-child transmission of HIV infection. We aimed to detect whether TFV and/or its prodrug, tenofovir disoproxil fumarate (TDF), are substrates of ATP-binding cassette (ABC) transporters that are functionally expressed in the placenta, namely P-glycoprotein (ABCB1/MDR1), Breast Cancer Resistance Protein (ABCG2/BCRP) and Multidrug Resistance-Associated Protein 2 (ABCC2/MRP2). We employed in-vitro cell-based assays and in-situ animal model to assess possible role of the efflux transporters in transplacental pharmacokinetics of TFV and TDF. In-vitro transport assays were performed in MDCKII cells transduced with human ABCB1, ABCG2 or ABCC2. To quantify the effect of these transporters on TFV/TDF transplacental passage, we employed the in-situ model of dually perfused rat term placenta in open and closed setup. In-vitro assays revealed that TDF is a dual substrate of ABCB1 and ABCG2 but not of ABCC2. In contrast, TFV transport was not influenced by any of these transporters. Applying concentration-dependent studies and selective inhibitors, we further confirmed these findings in situ on the organ level; both ABCB1 and ABCG2 limited mother-to-fetus transfer of TDF whereas TFV transplacental passage was not affected by these ABC transporters. We propose limited mother-to-fetus transport of both TFV and TDF. While placental transport of TFV is restricted passively, by physical-chemical properties of the molecule, mother-to-fetus passage of TDF is actively hindered by placental ABCB1 and ABCG2 transporters, pumping this compound from trophoblast back to maternal circulation.

Potent and specific inhibition of the biological activity of the type-II transmembrane serine protease matriptase by the cyclic microprotein MCoTI-II

Matriptase is a type-II transmembrane serine protease involved in epithelial homeostasis in both health and disease, and is implicated in the development and progression of a variety of cancers. Matriptase mediates its biological effects both via as yet undefined substrates and pathways, and also by proteolytic cleavage of a variety of well-defined protein substrates, several of which it shares with the closely-related protease hepsin. Development of targeted therapeutic strategies will require discrimination between these proteases. Here we have investigated cyclic microproteins of the squash Momordica cochinchinensis trypsin-inhibitor family (generated by total chemical synthesis) and found MCoTI-II to be a high-affinity (Ki 9 nM) and highly selective (> 1,000-fold) inhibitor of matriptase. MCoTI-II efficiently inhibited the proteolytic activation of pro-hepatocyte growth factor (HGF) by matriptase but not by hepsin, in both purified and cell-based systems, and inhibited HGF-dependent cell scattering. MCoTI-II also selectively inhibited the invasion of matriptase-expressing prostate cancer cells. Using a model of epithelial cell tight junction assembly, we also found that MCoTI-II could effectively inhibit the re-establishment of tight junctions and epithelial barrier function in MDCK-I cells after disruption, consistent with the role of matriptase in regulating epithelial integrity. Surprisingly, MCoTI-II was unable to inhibit matriptase-dependent proteolytic activation of prostasin, a GPI-anchored serine protease also implicated in epithelial homeostasis. These observations suggest that the unusually high selectivity afforded by MCoTI-II and its biological effectiveness might represent a useful starting point for the development of therapeutic inhibitors, and further highlight the role of matriptase in epithelial maintenance.

Evaluation of the Potency of Telaprevir and Its Metabolites as Inhibitors of Renal Organic Cation Transporters, a Potential Mechanism for the Elevation of Serum Creatinine

Telaprevir-based triple therapy is a highly effective treatment for chronic hepatitis C. However, adverse reactions include reversible and dose-dependent elevation of serum creatinine levels. We speculated that this effect reflects inhibition of the renal organic cation transporters hOCT2, hMATE1, and hMATE2-K by telaprevir or its metabolites (VRT-127394 and VRT-0922061). Telaprevir, VRT-127394, and VRT-0922061 showed negligible or weak effects on hOCT2 at concentrations of ≥20 µM, but inhibited hMATE1 by 35, 38, and 53% and hMATE2-K by 47, 45, and 61% at 100 µM, respectively. Telaprevir or its metabolites (10 µM) did not affect basal-to-apical transport of MPP(+) across monolayers of hOCT2-hMATE1 double-transfected MDCKII cells, whereas pyrimethamine, a potent inhibitor of hMATE1, markedly inhibited MPP(+) transport. Taken together, inhibition of hOCT2, hMATE1, and hMATE2-K is unlikely to be clinically relevant because unbound plasma concentrations of telaprevir and its metabolites reach only 2 µM following oral administration of a dose of 750 mg telaprevir. Hence, elevated serum creatinine during telaprevir therapy may not be related to direct inhibition of renal organic cation transporters.

Active elimination of the marine biotoxin okadaic acid by P-glycoprotein through an in vitro gastrointestinal barrier

The consumption of okadaic acid (OA) contaminated shellfish can induce acute toxic symptoms in humans such as diarrhea, nausea, vomiting and abdominal pain; carcinogenic and embryotoxic effects have also been described. Toxicokinetic studies with mice have shown that high cytotoxic doses of OA can pass the gastrointestinal barrier presumably by paracellular passage. However, in vitro studies using human intestinal Caco-2 cell monolayers to represent the intestinal barrier have shown that at low-dose exposure OA is transported against a concentration gradient suggesting an active efflux mechanism. Since P-glycoprotein (P-gp) transports a wide variety of substrates, we investigated its possible influence on the observed elimination of OA. We used two different cellular transwell models: (i) Caco-2 cell monolayer endogenously expressing human P-gp and simulating the intestinal barrier and (ii) MDCK-II cell monolayer stably over-expressing P-gp. Our study demonstrates clearly that OA at non-cytotoxic concentrations passes the monolayer barrier only to a low degree, and that it is actively eliminated by P-gp over the apical membrane. Therefore, our in vitro data indicate that humans appear to have efficient defense mechanisms to protect themselves against low-dose contaminated shellfish by exhibiting a low bioavailability as a result of active elimination of OA by P-gp.

Phorbol 12-myristate 13-acetate inhibits P-glycoprotein-mediated efflux of digoxin in MDCKII-MDR1 and Caco-2 cell monolayer models

To investigate the effects of phorbol 12-myristate 13-acetate (PMA), a PKC activator, on P-glycoprotein-mediated efflux of digoxin in two cell transport models. Caco-2 cells, wild MDCKII cells (MDCKII-WT) and MDCKII cells transfected stably with human MDR1-gene encoding P-gp (MDCKII-MDR1) were examined. Cell viability was evaluated with MTT assay. Bidirectional transport of digoxin was evaluated in these cells. Intracellular ATP level was measured using ATP assay. P-gp ATPase activity was analyzed using a Pgp-Glo(TM) assay. PMA (10 μmol/L) did not reduce the viability of the 3 types of cells. In Caco-2 and MDCKII-MDR1 cell monolayers, PMA (1, 10 and 100 nmol/L) dose-dependently inhibited the basolateral to apical transport of digoxin, but did not change the apical to basolateral transport. In addition, PMA did not affect both the basolateral to apical and apical to basolateral transport of digoxin in MDCKII-WT cell monolayer. In agreement with the above results, PMA dose-dependently reduced intracellular ATP level and stimulated P-gp ATPase activity in both Caco-2 and MDCKII-MDR1 cells. Verapamil (a positive control, 100 μmol/L) caused similar inhibition on digoxin efflux as PMA did, whereas 4α-PMA (a negative control, 100 nmol/L) had no effect. PMA significantly inhibited P-gp-mediated efflux of digoxin in both Caco-2 and MDCKII-MDR1 cell monolayers via PKC activation.

Transport characteristics and transporter‐based drug–drug interactions of TM‐25659, a novel TAZ modulator

The in vitro metabolic stability and transport mechanism of TM-25659, a novel TAZ modulator, was investigated in human hepatocytes and human liver microsomes (HLMs) based on the preferred hepatobiliary elimination in rats. In addition, the in vitro transport mechanism and transporter-mediated drug-drug interactions were evaluated using oocytes and MDCKII cells overexpressing clinically important drug transporters. After a 1 h incubation in HLMs, 92.9 ± 9.5% and 95.5 ± 11.6% of the initial TM-25659 remained in the presence of NADPH and UDPGA, respectively. Uptake of TM-25659 readily accumulated in human hepatocytes at 37 ºC (i.e. 6.7-fold greater than that at 4 ºC), in which drug transporters such as OATP1B1 and OATP1B3 were involved. TM-25659 had a significantly greater basal to apical transport rate (5.9-fold) than apical to basal transport rate in the Caco-2 cell monolayer, suggesting the involvement of an efflux transport system. Further studies using inhibitors of efflux transporters and overexpressing cells revealed that MRP2 was involved in the transport of TM-25659. These results, taken together, suggested that TM-25659 can be actively influxed into hepatocytes and undergo biliary excretion without substantial metabolism. Additionally, TM-25659 inhibited the transport activities of OATP1B1 and OATP1B3 with IC50 values of 36.3 and 25.9 μm, respectively. TM-25659 (100 μm) increased the accumulation of the probe substrate by 160% and 213%, respectively, through the inhibition of efflux function of P-gp and MRP2. In conclusion, OATP1B1, OATP1B3, P-gp and MRP2 might be major transporters responsible for the pharmacokinetics and drug-drug interaction of TM-25659, although their contribution to in vivo pharmacokinetics needs to be further investigated.

Addition of amino acid moieties to lapatinib increases the anti‐cancer effect via amino acid transporters

Anti-cancer agents delivered to cancer cells often show multi-drug resistance (MDR) due to expulsion of the agents. One way to address this problem is to increase the accumulation of anti-cancer agents in cells via amino acid transporters. Thus, val-lapatinib and tyr-lapatinib were newly synthesized by adding valine and tyrosine moieties, respectively, to the parent anti-cancer agent lapatinib without stability issues in rat plasma. Val-lapatinib and tyr-lapatinib showed enhanced anti-cancer effects versus the parent lapatinib in various cancer cell lines, including human breast cancer cells (MDA-MB-231, MCF7) and lung cancer cells (A549), but not in non-cancerous MDCK-II cells. A glutamine uptake study revealed that both val-lapatinib and tyr-lapatinib, but not the parent lapatinib, inhibited glutamine transport in MDA-MB-231 and MCF7 cells, suggesting the involvement of amino acid transporters. In conclusion, val-lapatinib and tyr-lapatinib have enhanced anti-cancer effects, likely due to an increased uptake of the agents into cancer cells via amino acid transporters. The present data suggest that amino acid transporters may be an effective drug delivery target to increase the uptake of anti-cancer agents, leading to one method of overcoming MDR in cancer cells.

Olomoucine II, but Not Purvalanol A, Is Transported by Breast Cancer Resistance Protein (ABCG2) and P-Glycoprotein (ABCB1)

Purine cyclin-dependent kinase inhibitors have been recognized as promising candidates for the treatment of various cancers; nevertheless, data regarding interaction of these substances with drug efflux transporters is still lacking. Recently, we have demonstrated inhibition of breast cancer resistance protein (ABCG2) by olomoucine II and purvalanol A and shown that these compounds are able to synergistically potentiate the antiproliferative effect of mitoxantrone, an ABCG2 substrate. In this follow up study, we investigated whether olomoucine II and purvalanol A are transported by ABCG2 and ABCB1 (P-glycoprotein). Using monolayers of MDCKII cells stably expressing human ABCB1 or ABCG2, we demonstrated that olomoucine II, but not purvalanol A, is a dual substrate of both ABCG2 and ABCB1. We, therefore, assume that pharmacokinetics of olomoucine II will be affected by both ABCB1 and ABCG2 transport proteins, which might potentially result in limited accumulation of the compound in tumor tissues or lead to drug-drug interactions. Pharmacokinetic behavior of purvalanol A, on the other hand, does not seem to be affected by either ABCG2 or ABCB1, theoretically favoring this drug in the potential treatment of efflux transporter-based multidrug resistant tumors. In addition, we observed intensive sulfatation of olomoucine II in MDCKII cell lines with subsequent active efflux of the metabolite out of the cells. Therefore, care should be taken when performing pharmacokinetic studies in MDCKII cells, especially if radiolabeled substrates are used; the generated sulfated conjugate may largely contaminate pharmacokinetic analysis and result in misleading interpretation. With regard to chemical structures of olomoucine II and purvalanol A, our data emphasize that even drugs with remarkable structure similarity may show different pharmacokinetic behavior such as interactions with ABC transporters or biotransformation enzymes.

Effect of the drug transporters ABCG2, Abcg2, ABCB1 and ABCC2 on the disposition, brain accumulation and myelotoxicity of the aurora kinase B inhibitor barasertib and its more active form barasertib-hydroxy-QPA

We explored whether barasertib (AZD1152), a selective Aurora B kinase inhibitor, is a substrate for P-glycoprotein (Pgp, MDR1), breast cancer resistance protein (BCRP), and multidrug resistance protein 2 (MRP2) in vitro. Cell survival, drug transport, and competition experiments with barasertib pro-drug and the more active form of the drug (barasertib-hQPA) were performed using MDCKII (wild type, MDR1, BCRP, and MRP2) and LLCPK (wild type and MDR1) cells and monolayers, and Sf9-BCRP membrane vesicles. Moreover we tested whether P-gp and BCRP affect the oral pharmacokinetics, tissue distribution, and myelotoxicity of barasertib in vivo using Bcrp1(-/-)/Mdr1a/1b (-/-) (triple knockout) and wild type mice. In cell survival experiments expression of BCRP and MDR1 resulted in significant resistance to barasertib. In transwell experiments, barasertib-hQPA was transported by BCRP and MDR1 efficiently. In Sf9-BCRP membrane vesicles, both barasertib and barasertib-hQPA significantly inhibited the BCRP-mediated transport of methotrexate. In contrast, no active transport of barasertib by MRP2 was observed, and overexpression of MRP2 did not affect cytotoxicity of barasertib. In vivo, systemic exposure as well as bioavailability, brain penetration, kidney and liver distribution and myelotoxicity of barasertib-hQPA were statistically significantly increased in Bcrp1(-/-)/Mdr1a/1b(-/-) compared with wild type mice (p<0.001). Barasertib is transported efficiently by P-gp and BCRP/Bcrp1 in vitro. In vivo, genetic deletion of P-gp and BCRP in mice significantly affected pharmacokinetics, tissue distribution and myelotoxicity of barasertib-hQPA. Possible clinical consequences for the observed affinity of barasertib for P-gp and BCRP need to be explored.

Reply to the comment of Ana I. Alvarez and Gracia Merino regarding “Assessment of ABCG2-mediated transport of xenobiotics across the blood-milk barrier of dairy animals using a new MDCKII in vitro model” by Wassermann et al. 2013

June 2009) and GQ141082 (oABCG2: submitted on 3 July 2009). These generated ABCG2 full-length clones consist of the open reading frame (ORF) sequence as well as an 5′ untranslated region (5′-UTR) where regulatory elements are located. In this context, we identified besides other reg-ulatory elements such as SP-1 and AP-1 aryl hydrocarbon receptor response elements (AhRE) in the bovine as well as the caprine ABCG2 5′-UTR (Wassermann et2013 al. ). In further mechanistic studies, we showed that the func-tional expression of ABCG2 was induced due to binding of ligand-activated AhR to AhRE sequences in the ABCG2 5′-UTR (Halwachs et2012). Merino and coworkers al. (Merino et al. 2009) as well as Real and coworkers (Real et al. 2011) did not provide NCBI accession numbers for the hepatocellular ABCG2 cDNA sequences in their pub-lications. To our knowledge, the NCBI database entry (NM_001037478) from Real et al. was first published and accessible in June 2012. Since then, it was not possible to perform sequence analysis to determine cDNA sequence homology between the bovine hepatocellular and mam-mary ABCG2 clones. In addition, we did not only gener-ate a full-length bovine mammary ABCG2 cDNA sequence but also caprine and ovine mammary ABCG2 sequences. In this context, differences between the three species occurred regarding the ABCG2 cDNA as well as protein sequences (Wassermann etal. 2013 ). Former studies (Wassermann et al. 2013) as well as the present publication additionally revealed species-dependent differences regarding the sub-strate specificity of the generated MDCKII-ABCG2 cell lines. However, the aim of our research was to generate tis-sue-specific full-length ABCG2 cDNA clones to investigate the influence of xenobiotics on the tissue-specific expres-sion of ABCG2. In this context, the generated MDCKII cell lines stably expressing bABCG2, cABCG2 or oABCG2 represent a new in vitro model.

Innovative in Vitro Method To Predict Rate and Extent of Drug Delivery to the Brain across the Blood–Brain Barrier

The relevant parameters for predicting rate and extent of access across the blood-brain barrier (BBB) are fu,plasma (unbound fraction in plasma), Vu,brain (distribution volume in brain) and Kp,uu,brain (ratio of free concentrations in plasma and brain). Their estimation still requires animal studies and in vitro low throughput experiments which make difficult the screening of new CNS candidates. The aim of the present work was to develop a new whole in vitro high throughput method to predict drug rate and extent of access across the BBB. The system permits estimation of fu,plasma, Vu,brain and Kp,uu,brain in a single experimental system, using in vitro cell monolayers in different conditions. From the ratios of the apparent permeability values (Papp) with the adequate mathematical analysis the relevant parameters can be estimated. Papp of ten model compounds has been obtained in MDCKII and MDCK-Mdr1cell monolayers in the absence and presence of albumin and brain homogenate. The ratio of Papp in the absence and presence of albumin allows estimation of in vitro fu,plasma. Papp in the presence of brain homogenate is used to estimate fu,brain and Vu,brain. Kp,uu,brain is estimated from the apical to basal versus basal to apical clearances. The BBB parameters obtained with the new method were predictive of the in vivo behavior of candidates. In vitro fu,plasma, Kp,uu,brain and Vu,brain (calculated with Papp from MDCKII cell line) presented a good correlation with in vivo fu,plasma, Kp,uu,CSF and Vu,brain published values (r=0.92; r=0.85; and r=0.99 respectively). Despite its simplicity the predictive performance is fairly good considering the reduced number of tested compounds with different physicochemical and transport properties. Further experimental modifications could be checked to optimize the method, but the present data support its feasibility. As other in vitro cell culture models, the system is suitable for miniaturization and robotization to allow high throughput screening of CNS candidates.