Screening of Multidrug-Resistance Sensitive Drugs by In Situ Brain Perfusion in P-Glycoprotein-Deficient Mice

Purpose. This study was conducted to assess the influence of P-glycoprotein (P-gp) on brain uptake of multidrug resistance sensitive drugs using an in situbrain perfusion technique in P-gp-deficient (mdr1a[–/–]) and wild-type mice. Methods. The blood-brain transport of radiolabeled vinblastine, vincristine, doxorubicin, colchicine, and morphine was evaluated in mdr1a(–/–) and wild-type CF-1 mice with the in situ brain perfusion technique. Brain uptake of drugs after intravenous pretreatment with P-gp reversal agents, (PSC 833, GF 120918, or (±)-verapamil), or vehicle also was studied in wild-type mice. In all experiments, cerebral vascular volume was determined by co-perfusion of sucrose. Results. Cerebral vascular volume was preserved during perfusion, indicating maintenance of blood-brain barrier integrity in both types of mice within the concentration range of substrates in the perfusate. The apparent brain transport of colchicine, vinblastine, doxorubicin, and morphine was increased 3.0, 2.7, 1.5, and 1.4-fold, respectively, in mdr1a(–/–) mice compared with the wild-type; the brain uptake of vincristine was not affected by P-gp. Preadministration of PSC 833 or GF 120918 in wild-type mice led to a 3-fold increase in the brain transport of colchicine and vinblastine, but no effect was observed for the other compounds. Intravenous verapamil enhanced colchicine brain transport (1.8-fold), but failed to increase the brain uptake of vinblastine and morphine. Conclusion. The in situ brain perfusion technique appears to be a sensitive and powerful tool for medium throughput screening of the brain uptake of multidrug resistance sensitive drugs. The effect of P-gp is characterized more efficiently with mdr1a(–/–) mice than by using modulators of P-gp in wild-type mice.     

Astrocytes Increase the Functional Expression of P-Glycoprotein in an In Vitro Model of The Blood-Brain Barrier

Purpose. To investigate the influence of astrocytes on P-glycoprotein (Pgp) expression and intracellular accumulation of Pgp substrates, separate from their net transcellular transport across the blood-brain barrier (BBB). Methods. An in vitroBBB model was used, comprising of brain capillary endothelial cells (BCEC) monolayers or BCEC co-cultured with astrocytes. Results. BCEC+astrocyte co-cultures seemed to express a higher level of Pgp compared to BCEC monolayers. Inhibition of Pgp results in an increased intracellular accumulation of Pgp substrates in both BCEC monolayers and BCEC+astrocyte co-cultures, and increased the sensitivity for vinblastine mediated disruption of the in vitro BBB (called the vinblastine exclusion assay). BCEC monolayers were more sensitive to vinblastine mediated disruption compared to BCEC+astrocyte co-cultures. In the latter, but not in BCEC monolayers, an inhibitable polar transport of Pgp substrates was only found from the brain to the blood side of the filter. Conclusions. Astrocytes increase the functional expression of Pgp in our in vitroBBB model. These results also illustrate that an important role for Pgp on the BBB is to protect the barrier against intracellular accumulation of cytotoxic BBB disrupting compounds.     

In Vivo Saturation of the Transport of Vinblastine and Colchicine by P-Glycoprotein at the Rat Blood–Brain Barrier

Purpose. To determine concentration-dependent P-gp-mediated efflux across the luminal membrane of endothelial cells at the blood-brain barrier (BBB) in rats. Methods. The transport of radiolabeled colchicine and vinblastine across the rat BBB was measured with or without PSC833, a well known P-gp inhibitor, and within a wide range of colchicine and vinblastine concentration by an in situ brain perfusion. Thus, the difference of brain transport achieved with or without PSC833 gives the P-gp-mediated efflux component of the compound transported through the rat BBB. Cerebral vascular volume was determined by coperfusion with labeled sucrose in all experiments. Results. Sucrose perfusion indicated that the vascular space was close to normal in all the studies, indicating that the BBB remained intact. P-gp limited the uptake of both colchicine and vinblastine, but the compounds differ in that vinblastine inhibited its own transport. Vinblastine transport was well fitted by a Hill equation giving IC₅₀ at 71 M, a Hill coefficient (n) 2, and a maximal efflux velocity J_max of 9 pmol s^–1 g^–1 of brain. Conclusions. P-gp at the rat BBB may carry out both capacity-limited and capacity-unlimited transport, depending on the substrate, with pharmacotoxicologic significance for drug brain disposition and risk of drug-drug interactions.     

Inhibition of P-Glycoprotein by D-α-Tocopheryl Polyethylene Glycol 1000 Succinate (TPGS)

Purpose. To investigate whether d--tocopheryl polyethylene glycol 1000 succinate (TPGS) functions as an inhibitor of P-glycoprotein (P-gp), the multidrug resistance transporter. Methods. Two assays were used to measure the function of TPGS on P-gp function. First, we examined the ability of TPGS to modulate the cytotoxicity of established, cytotoxic, P-glycoprotein substrates. Parental NIH 3T3 cells and NIH 3T3 cells transfected with the human MDR1 cDNA (G185) were exposed to doxorubicin, paclitaxel, colchicine, vinblastine and 5-fluorouracil (5FU) in the presence or absence of TPGS. Cytotoxicity was assessed with the MTT assay. Second, polarized transport of the P-gp substrates rhodamine 123 (R123), paclitaxel and vinblastine was measured using the human intestinal HCT-8 and Caco-2 cell lines grown in Transwell dishes. Drug flux was measured by liquid scintillation counting or fluorescence spectroscopy of the media. Results. G185 cells were 27–135 fold more resistant to the cytotoxic drugs doxorubicin, vinblastine, colchicine and paclitaxel than the parental NIH 3T3 cells. In contrast 5FU, which is not a P-gp substrate, is equally cytotoxic to parental and G185 cells. Co-administration of TPGS enhanced the cytotoxicity of doxorubicin, vinblastine, paclitaxel, and colchicine in the G185 cells to levels comparable to the parental cells. TPGS did not increase the cytotoxicity of 5FU in the G185 cells. Using a polarized epithelial cell transport assay, TPGS blocked P-gp mediated transport of Rl 23 and paclitaxel in a dose responsive manner. Conclusions. These data demonstrate that TPGS acts as a reversal agent for P-glycoprotein mediated multidrug resistance and inhibits P-gp mediated drug transport. These results suggest that enhanced oral bioavailability of drugs co-administered with TPGS may, in part, be due to inhibition of P-glycoprotein in the intestine.     

MDCK Cell Cultures as an Epithelial In Vitro Model: Cytoskeleton and Tight Junctions as Indicators for the Definition of Age-Related Stages by Confocal Microscopy

Purpose. Madin Darby Canine Kidney (MDCK) cells were grown in culture, and age-related morphological changes in the cytoskeleton and tight junction (TJ) network were used to define stages in view of establishing an optimal in vitro model for the epithelial barrier. Methods. Growth curves and transepithelial electrical resistance (TEER) were determined, and the cytoskeleton (actin, -tubulin, vimentin) and TJ (Zonula occludens proteins ZO1, ZO2) were investigated with immunofluorescent methods by confocal laser scanning microscopy (CLSM) and digital image restoration. Results. TEER measurements indicated that TJ were functional after one day. Values then remained constant. Four morphological stages could be distinguished. Stage I (0–1 day): Sub confluent cultures with flat cells; TJ established after cell-to-cell contacts are made. Stage II (2–6 days): Confluent monolayers with a complete TJ network, which remains intact throughout the later stages. Stage III (7–14 days): Rearrangement in the cytoskeleton; constant cell number; volume and surface area of cells reduced (cobble-stone appearance). Stage IV ( 15 days): Dome formation, i.e. thickening and spontaneous uplifting of the cell monolayer. Conclusions. Based on the structural characteristics of stage III cell cultures, which are closest to the in vivo situation, we expect them to represent an optimal in vitromodel to study drug transport and/or interactions with drugs and excipients.     

P-Glycoprotein Is More Efficient at Limiting Uptake than Inducing Efflux of Colchicine and Vinblastine in HL-60 Cells

Purpose. To investigate the role of the P-glycoprotein (P-gp) drug efflux pump in the intracellular disposition of colchicine and vinblastine. Methods. Uptake and efflux kinetics were studied in vitro in human lymphocytes and in HL-60 cells with or without the P-gp modulator, verapamil. Results. In human lymphocytes, colchicine was slowly taken up (uptake half-life was 18.9 ± 1.1 hr.) and verapamil increased colchicine uptake by 37%, whereas it did not modify colchicine efflux from cells. In HL-60 cells, colchicine uptake was non-linear and slower than that of vinblastine, the colchicine uptake half-life (11.1 ± 0.5 hr.) being 25-fold longer than that of vinblastine at 25 nM. Verapamil did not significantly modify colchicine uptake half-life, but increased its intracellular accumulation by 23% and that of vinblastine by 81%. Immuno-flow cytometry showed that P-gp expression in HL-60 cells increased significantly from 24 hr. following colchicine or vinblastine exposure. The significant increase in colchicine uptake induced by verapamil at 24 hr. was correlated with this enhanced P-gp expression. The drug efflux half-life was 11.5-fold higher for colchicine (23 ± 0.9 hr) than vinblastine, indicating a much slower elimination of colchicine from cells that could be related to its longer dissociation half-life from the tubulin receptor. Verapamil treatment did not modulate either colchicine or vinblastine efflux kinetics, suggesting that the intracellular drugs are not available to the transmembrane P-gp binding sites. Conclusions. P-gp may not be the main reason for the slowness of colchicine uptake. It may be more efficient at controlling entry of colchicine and vinblastine through the plasma membrane than at mediating their efflux from HL-60 cells.     

Apparent Lack of Mrp1-Mediated Efflux at the Luminal Side of Mouse Blood-Brain Barrier Endothelial Cells

Purpose. The purpose of this work was to determine mrp1-mediated efflux across the luminal membrane of endothelial cells at the blood-brain barrier (BBB) in mice. Methods. The transport of radiolabeled etoposide, 17-estradiol-D-17-glucuronide (E₂17G), vincristine, and doxorubicin across the BBB of mrp1(–/–) and wild-type mice was evaluated by in situ brain perfusion. Etoposide transport was also determined in P-glycoprotein-deficient mdr1a(–/–) mice perfused with both etoposide and mrp1 inhibitors like probenecid or MK571. Cerebral vascular volume was determined by co-perfusion with labeled sucrose. Results. Sucrose perfusion indicated that the vascular space was close to normal in all the studies, indicating that the BBB remained intact. The transport of etoposide, E₂17G, vincristine, and doxorubicin into the brain was not affected by the lack of mrp1. Trans-efflux studies in mrp1-deficient mice with etoposide and E₂17G confirmed that mrp1 was not involved in the efflux of these substrates across the BBB. There was also a significant P-gp-mediated efflux of etoposide in studies with P-glycoprotein-deficient mdr1a(–/–) mice. Perfusion of mdr1a(–/–) mice etoposide plus probenecid or MK571 did not affect the brain transport of etoposide. Conclusion. Efflux mediated by mrp1 does not seem to occur across the luminal membrane of the endothelial cells forming the mouse BBB.     

BBB Transport and P-glycoprotein Functionality Using MDR1A (-/-) and Wild-Type Mice. Total Brain Versus Microdialysis Concentration Profiles of Rhodamine-123

Purpose. The effect of P-glycoprotein (Pgp) on brain distribution using mdrla (-/-) mice was investigated. Methods. Fluorescein (Flu) and FD-4 were used to check whether blood-brain barrier (BBB) integrity was maintained in mdrla (-/-) mice. The Pgp substrate rhodamine-123 (R123) was infused and total brain, blood and brain microdialysate concentrations in mdrla (-/-) mice and wild-type mice were compared. Results. Maintenance of BBB integrity was indicated by equal total brain/blood ratios of Flu and FD-4 in both mice types. R123 concentrations in brain after i.v. infusion were about 4-fold higher in mdrla (-/-) than in wild-type mice (P < 0.05), without changes in blood levels. After microdialysis experiments the same results were found, excluding artifacts in the interpretation of Pgp functionality by the use of this technique. However the 4-fold ratio in brain was not reflected in corresponding microdialysates. No local differences of R123 in the brain were found. By the no-net-flux method in vivo recovery appeared to 4.6-fold lower in mdrla (-/-) mice compared with wild-type mice. Conclusions. Pgp plays an important role in R123 distribution into the brain. Using intracerebral microdialysis, changes in in vivo recovery by the absence or inhibition of Pgp (or active efflux in general) need to be considered carefully.     

The Application of Bovine Brain Microvessel Endothelial-Cell Monolayers Grown onto Polycarbonate Membranes in Vitro to Estimate the Potential Permeability of Solutes Through the Blood–Brain Barrier

Previously our laboratory (Rim et aL, Int. J. Pharm. 32:79–84, 1986) described an in vitro blood-brain barrier (BBB) model consisting of cultured bovine brain microvessel endothelial cells (BMECs) grown onto regenerated cellulose acetate membranes. However, the utility of this in vitro BBB model system was limited because the regenerated cellulose acetate membrane and not the monolayer of bovine BMECs was rate limiting for the permeability of very lipophilic compounds. Therefore, in this study we have evaluated polycarbonate membranes as supports for growing bovine BMECs and for conducting in vitro drug permeability studies. Bovine BMECs were cultured on collagen-coated polycarbonate membranes (13-mm diameter, 12-µm pore size) which were then mounted into side-by-side diffusion cells for transport studies. The permeabilities of a series of solutes of varying lipophilicity (progesterone, estrone, testosterone, haloperidol, propranolol, antipyrine, caffeine, urea, acyclovir, ganciclovir, ribavirin, and glycerol) were determined and an excellent correlation (r = 0.97) was established between the permeability coefficients of the solutes and their log partition coefficients (PC)/(MW)^1/2. These results suggest that bovine BMECs cultured onto polycarbonate membranes can be used as an in vitro model system for estimating the potential permeability of a solute through the BBB in vivo.     

Hydrogen Bonding Potential as a Determinant of the in Vitro and in Situ Blood–Brain Barrier Permeability of Peptides

With the exception of various central nervous system (CNS)-required nutrients for which specific, saturable transport systems exist, the passage of most water-soluble solutes through the blood–brain barrier (BBB) is believed to depend largely on the lipid solubility of the solutes. Most peptides, therefore, do not enter the CNS because of their hydrophilic character. Recently, utilizing homologous series of model peptides and Caco-2 cell monolayers as a model of the intestinal mucosa, it was concluded that the principal determinant of peptide transport across the intestinal cellular membrane is the energy required to desolvate the polar amide bonds in the peptide (P. S. Burton et al., Adv. Drug Deliv. Rev. 7:365–386, 1991). To determine whether this correlation can be extended to the BBB, the permeabilities of the same peptides were determined using an in vitro as well as an in situ BBB model. The peptides, blocked on the N- and C-terminal ends, consisted of D-phenylalanine (F) residues: AcFNH₂, AcF₂NH₂, AcF₃NH₂, AcF₂(NMeF)NH₂, AcF(NMeF)₂NH₂, Ac(NMeF)₃NH₂, and Ac(NMeF)₃NHMe. A good correlation among the permeabilities of these model peptides across the bovine brain microvessel endothelial cell (BBMEC) monolayers, an in vitro model of the BBB, and their permeabilities across the BBB in situ was observed (r = 0.928, P < 0.05). The permeabilities of these peptides did not correlate with the octanol–buffer partition coefficients of the peptides (r = 0.389 in vitro and r = 0.155 in situ; P < 0.05). However, correlations were observed between the permeabilities of these peptides and the number of potential hydrogen bonds the peptides can make with water (r = 0.837 in vitro and r = 0.906 in situ; P < 0.05), suggesting that desolvation of the polar bonds in the molecule is a determinant of permeability. Consistent with this, good correlations were found between the permeabilities of these peptides and their partition coefficients between heptane–ethylene glycol (r = 0.981 in vitro and r = 0.940 in situ ; P < 0.05) or the differences in partition coefficients between octanol–buffer and isooctane–buffer (logPC) (r = 0.961 in vitro and r = 0.962 in situ; P < 0.05), both of which are experimental estimates of hydrogen bond or desolvation potential. These results suggest that the permeability of peptides through the BBB is governed by the same physicochemical parameter (hydrogen bonding potential) as their permeability through the intestinal mucosa.     

Transport of Levofloxacin in the OK Kidney Epithelial Cell Line: Interaction with p-Aminohippurate Transport

Purpose. To evaluate the mechanism of renal transport of quinolone antibacterial drugs, we examined the interaction of levofloxacin with p–aminohippurate (PAH) transport systems and the transport of levofloxacin in renal epithelial cells. Methods. Transport of [¹⁴C]PAH or [¹⁴C]levofloxacin was measured using OK cell monolayers grown on microporous membrane filters. Results. Transcellular transport from the basolateral to the apical side and cellular accumulation of [¹⁴C]PAH were inhibited by levofloxacin. Both the initial uptake of [¹⁴C]PAH from the basolateral side and the efflux to the apical side were inhibited by levofloxacin. The basolateral–to–apical transcellular transport of [¹⁴C]levofloxacin was greater than that in the opposite direction. [¹⁴C]Levofloxacin efflux to the apical side was greater than that to the basolateral side. Unlabeled levofloxacin and grepafloxacin inhibited the transcellular transport of [¹⁴C]levofloxacin, accompanied by an increase of cellular accumulation. However, neither PAH nor an anion transport inhibitor 4–4–diisothiocyanostilbene–2,2–disulfonic acid (DIDS) affected the basolateral–to–apical transport of [¹⁴C]levofloxacin nor its uptake from the basolateral side. Conclusions. These results indicated that levofloxacin inhibits PAH transport across both the basolateral and apical membranes of OK cells, but are not transported via the systems for PAH transport. The existence of a specific transport system for quinolones was indicated in OK cells.     

Characteristics of Choline Transport Across the Blood-Brain Barrier in Mice: Correlation with In Vitro Data

Purpose. We examined the functional properties of choline transport across the blood-brain barrier (BBB) in mice. We compared the kinetic parameters and transport properties with those found in our in vitro uptake experiments using mouse brain capillary endothelial cells (MBEC4). Methods. The permeability coefficient-surface area product (PS) values of [³H]choline at the BBB were estimated by means of anin situ brain perfusion technique in mice.Results. [³H]Choline uptake was well described by a two-component model: a saturable component and a nonsaturable linear component. The [³H]choline uptake was independent of pH and Na⁺, but was significantly decreased by the replacement of Na⁺ with K⁺. Various basic drugs, including substrates and inhibitors of the organic cation transporter, significantly inhibited the [³H]choline uptake. These in situ (in vivo) results corresponded well to the in vitro results and suggest that the choline transporter at the BBB is a member of the organic cation transporter (OCT) family. Conclusion. The choline transport mechanism at the BBB is retained in MBEC4.     

The Effect of Cetylpyridinium Chloride (CPC) on the Cell Surface Hydrophobicity and Adherence of Candida albicansto Human Buccal Epithelial Cells in Vitro

Purpose. This study examined the effects of cetylpyridinium chloride (CPC) on cell surface hydrophobicity (CSH) and adherence of blastospores of Candida albicans(MEN strain) to human buccal epithelial cells (EEC) in vitro. Methods. The effect of CPC treatment of either C. albicans blastospores or BEC on their subsequent adherence was determined using ³⁵SO₄ labelled blastospores in association with a Percoll gradient. The effects of CPC treatment of blastospores on their CSH was determined using Hydrophobic Interaction Chromatography. Results. Treatment of exponential and stationary phase blastospores with CPC (50 µg mL^–1) for 0.5–30 minutes, or with CPC (0.5–50 µg mL^–1) for 15 minutes resulted in significant reductions in both blastospore CSH and adherence to BEC in vitro. No correlation was apparent (r < 0.8) between reduced CSH and reduced blastospore adherence following treatment with CPC (0.5–50 µg mL^–1). Significantly reduced adherence of C. albicans (stationary or exponential growth phases) to human EEC was also observed following treatment of BEC with CPC (50 µg mL^–1) for 0.5–30 minutes or with CPC (0.5–50 µg mL^–1) for 15 minutes. Antiadherence effects were observed at both sub and super-minimum inhibitory concentrations of CPC. Conclusions. It is suggested that, whilst the ability of CPC to reduce the CSH of C. albicans may contribute to its reduced adherence to human BEC in vitro, reduced CSH is only one of several possible factors that contribute to the observed antiadherence effects.     

The association of statins plus LDL receptor-targeted liposome-encapsulated doxorubicin increases in vitro drug delivery across blood–brain barrier cells

BACKGROUND AND PURPOSE

The passage of drugs across the blood–brain barrier (BBB) limits the efficacy of chemotherapy in brain tumours. For instance, the anticancer drug doxorubicin, which is effective against glioblastoma in vitro, has poor efficacy in vivo, because it is extruded by P-glycoprotein (Pgp/ABCB1), multidrug resistance-related proteins and breast cancer resistance protein (BCRP/ABCG2) in BBB cells. The aim of this study was to convert poorly permeant drugs like doxorubicin into drugs able to cross the BBB.

EXPERIMENTAL APPROACH

Experiments were performed on primary human cerebral microvascular endothelial hCMEC/D3 cells, alone and co-cultured with human brain and epithelial tumour cells.

KEY RESULTS

Statins reduced the efflux activity of Pgp/ABCB1 and BCRP/ABCG2 in hCMEC/D3 cells by increasing the synthesis of NO, which elicits the nitration of critical tyrosine residues on these transporters. Statins also increased the number of low-density lipoprotein (LDL) receptors exposed on the surface of BBB cells, as well as on tumour cells like human glioblastoma. We showed that the association of statins plus drug-loaded nanoparticles engineered as LDLs was effective as a vehicle for non-permeant drugs like doxorubicin to cross the BBB, allowing its delivery into primary and metastatic brain tumour cells and to achieve significant anti-tumour cytotoxicity.

CONCLUSIONS AND IMPLICATIONS

We suggest that our ‘Trojan horse’ approach, based on the administration of statins plus a LDL receptor-targeted liposomal drug, might have potential applications in the pharmacological therapy of different brain diseases for which the BBB represents an obstacle.     

Carrier-Mediated Transport of Baclofen Across Monolayers of Bovine Brain Endothelial Cells in Primary Culture

The mechanism of transport of baclofen, a centrally acting muscle relaxant, across the blood–brain barrier (BBB) was studied using an in vitro model. The model consisted of a monolayer of bovine brain endothelial cells grown in primary culture on a porous regenerated cellulose membrane. The transport of baclofen across the monolayer expressed time and concentration dependency and was saturable. Transport data were corrected for diffusion and fitted to the Michaelis–Menten V _max model: K _m = 58.5 µM, V _max = 0.23 nmol/min. The results validate the use of the in vitro BBB model as described and support the hypothesis that baclofen penetrates the BBB by means of a carrier-mediated transport system.     

Mdr1 transfection causes enhanced apoptosis by paclitaxel: an effect independent of drug efflux function of P-glycoprotein

Purpose. We previously reported that in patient tumors the expression of the mdr1 p-glycoprotein (Pgp) resulted in a lower paclitaxel-induced inhibition of DNA precursor incorporation, but a higher apoptosis (Clin. Cancer Res. 4:2949-2955, 1998). The present study was to evaluate these findings in an experimental system where the Pgp effect can be studied without confounding factors such as the intra- and inter-tumor heterogeneity associated with patient tumors. Methods. To separate the effect of Pgp on intracellular paclitaxel accumulation from its effects on drug sensitivity, we compared the drug activity at various extracellular and intracellular drug concentrations using the human breast MCF7 tumor cells and its mdr1-transfected variant BC19 cells. Results. Compared to MCF7 cells, BC19 cells showed a 9-fold higher Pgp level and >13-fold higher mdr1 expression. Intracellular paclitaxel accumulation was 80-130% lower in BC19 cells when the extracellular concentrations were 100 nM, but the difference was reduced to <15% differences at higher extracellular concentrations of 1,000 nM. For the G2/M block effect MCF7 cells were 43-fold more sensitive than BC19 cells at equal extracellular concentration, and 3.5-fold more sensitive at comparable intracellular concentrations. On the contrary, BC19 cells were more sensitive to the apoptotic effect; BC19 cells showed equal or higher apoptosis compared to MCF7 cells at extracellular concentrations above 100 nM, and a 30-100% higher apoptosis at comparable intracellular concentrations. Conclusions. These results confirm our previous observations in patient tumors and indicate that enhanced Pgp expression is associated with enhanced sensitivity to the apoptotic effect of paclitaxel and reduced sensitivity to its G2/M block effect, via yet-unknown mechanisms that are unrelated to the effect of Pgp on intracellular drug accumulation.     

Kinetic Analysis of P-Glycoprotein-Mediated Transport by Using Normal Human Placental Brush-Border Membrane Vesicles

Purpose. P-Glycoprotein (Pgp) plays an important role in drug disposition and excretion in various tissues such as the brain, intestine, and kidney. Moreover, we have demonstrated that Pgp is expressed on the brush-border membranes of trophoblast cells in the placenta and restricts drug transfer from the maternal circulation to the fetus. However, the transport kinetics of physiologically expressed Pgp has scarcely been investigated. Methods. In this study, we assessed the functional kinetics of transport mediated by Pgp that is physiologically expressed in normal tissue by using human placental brush-border membrane vesicles (BBMVs). Digoxin and vinblastine were used as typical substrates of Pgp. Results. The uptakes of [³H]digoxin and [³H]vinblastine into BBMVs were significantly increased in the presence of an ATP-regenerating system. The ATP-dependent uptakes of [³H]digoxin and [³H]vinblastine into BBMVs exhibited saturable kinetics. The Michaelis constants (K _t values) were 2.65 ± 1.80 M and 21.9 ± 3.37 M, respectively. In the presence of a Pgp inhibitor such as verapamil, cyclosporine A, or progesterone, the ATP-dependent uptakes of [³H]digoxin and [³H]vinblastine into BBMVs were significantly reduced. Anti-Pgp monoclonal antibody C219 completely inhibited the uptake of [³H]digoxin. Conclusions. The transport kinetics of [³H]digoxin and [³H]vinblastine by physiologically expressed Pgp were successfully evaluated by using BBMVs prepared from normal human placenta. The present method enabled us to evaluate the function of physiologically expressed Pgp and is superior to the use of cultured transfectants in terms of the yield of vesicles. The present method may also be applicable to investigating the influence of various factors such as the genotype of the MDR1 gene or various pathophysiologic states of neonates on the function of Pgp.     

Effect of mdr1a P-glycoprotein gene disruption, gender, and substrate concentration on brain uptake of selected compounds

Purpose. This study assessed the influence of mdr1a P-glycoprotein (P-gp) gene disruption, gender and concentration on initial brain uptake clearance (Cl _up) of morphine, quinidine and verapamil. Methods. Cl _up of radiolabeled substrates was determined in P-gp-competent and deficient [mdr1a(–/–)] mice by in situ brain perfusion. Brain:plasma distribution of substrates after i.v. administration was determined in both strains. Results. Genetic disruption of mdr1a P-gp resulted in 1.3-, 6.6- and 14-fold increases in Cl _up for morphine, verapamil and quinidine, respectively. With the exception of small differences for verapamil, gender did not affect Cl _up. Saturable transport of verapamil and quinidine was observed only in P-gp-competent mice, with apparent IC ₅₀ values for efflux of 8.6 ± 2.3 M and 36 ± 2 M, respectively. VerapamilCl _up was 50% higher in mdr1a(+/–) vs. mdr1a(+/+) mice; no such difference was observed for quinidine. In P-gp-competent mice, uptake of verapamil and quinidine was unaffected by organic vehicles. Plasma decreased VER Cl _up to a greater extent in the presence of P-gp. The influence of P-gp in situ was lower than, but correlated with, the effect in vivo. Conclusions. P-gp decreases Cl _up of morphine, verapamil and quinidine in situ with little or no influence of gender, but this effect cannot fully account for the effects of P-gp in vivo. P-gp is the only saturable transport mechanism for verapamil and quinidine at the murine blood-brain barrier. The influence of protein binding on Cl _up may be enhanced by P-gp-mediated efflux.     

Simultaneous in Vitro Measurement of Intestinal Tissue Permeability and Transepithelial Electrical Resistance (TEER) Using Sweetana–Grass Diffusion Cells

A simple modification of the commercially available Sweetana–Grass (S-G) side-by-side diffusion cells, allowing the simultaneous measurement of tissue permeability and transepithelial electrical resistance (TEER), has been described and validated for rat excised, muscle-free intestinal tissue. The TEER-lowering effects of a series of acylcarnitines were shown to be correlated with previously reported in vitro (i.e., membrane perturbation) and in vivo (i.e., absorption enhancement) activity. The TEER-lowering effect of palmitoyl carnitine chloride (PCC) was also shown to be reversible. The effects of PCC on TEER and the permeability of poorly absorbed compounds (cefoxitin and lucifer yellow) were simultaneously determined. Compared to controls (mannitol-treated), PCC immediately produced a rapid drop in colon TEER. By 5 min post-PCC addition, colon TEER was 50% of control; by 10 min post-PCC addition, colon TEER was 17% of control. After a lag of about 5–10 min post-PCC addition, the cefoxitin or lucifer yellow permeability coefficient increased more than 20-fold. The modified S-G cells provide a simple and reproducible method whereby flux and TEER can be simultaneously determined, providing a valuable link between the effect of absorption enhancers on TEER measurements and the increased permeability of poorly absorbed compounds.