Examination of blood-brain barrier (BBB) integrity in a mouse brain tumor model.

The present study evaluates, both functionally and biochemically, brain tumor-induced alterations in brain capillary endothelial cells. Brain tumors were induced in Balb/c mice via intracranial injection of Lewis Lung carcinoma cells into the right hemisphere of the mouse brain using stereotaxic apparatus. Blood-brain barrier (BBB) permeability was assessed at various stages of tumor development, using both radiolabeled tracer permeability and magnetic resonance imaging with gadolinium diethylene-triamine-pentaacetate contrast enhancement (Gad-DTPA). The expression of the drug efflux transporter, P-glycoprotein (P-gp), in the BBB at various stages of tumor development was also evaluated by Western blot and immunohistochemistry. Median mouse survival following tumor cell injection was 17 days. The permeability of the BBB to (3)H-mannitol was similar in both brain hemispheres at 7 and 10 days post-injection. By day 15, there was a twofold increase in (3)H-mannitol permeability in the tumor bearing hemispheres compared to the non-tumor hemispheres. Examination of BBB permeability with Gad-DTPA contrast enhanced MRI indicated cerebral vascular permeability changes were confined to the tumor area. The permeability increase observed at the later stages of tumor development correlated with an increase in cerebral vascular volume suggesting angiogenesis within the tumor bearing hemisphere. Furthermore, the Gad-DPTA enhancement observed within the tumor area was significantly less than Gad-DPTA enhancement within the circumventricular organs not protected by the BBB. Expression of P-gp in both the tumor bearing and non-tumor bearing portions of the brain appeared similar at all time points examined. These studies suggest that although BBB integrity is altered within the tumor site at later stages of development, the BBB is still functional and limiting in terms of solute and drug permeability in and around the tumor.

Induction of drug efflux protein expression by venlafaxine but not desvenlafaxine.

Venlafaxine and its metabolite desvenlafaxine are serotonin-norepinephrine reuptake inhibitors currently prescribed for the treatment of depression. Previously, it was reported that venlafaxine is an inducer of MDR1, the gene responsible for P-glycoprotein (P-gp). The present study expanded upon these findings by examining the effect of venlafaxine and desvenlafaxine on the expression of both P-gp and the breast cancer resistance protein (BCRP) in human brain endothelial cells (HBMEC), an in vitro model of the blood-brain barrier (BBB). The HBMEC were treated for 1 h with various concentrations (500 nM to 50 µM) of venlafaxine and desvenlafaxine. Western blot analysis revealed treatment with venlafaxine significantly induced the expression of P-gp (2-fold) and BCRP (1.75-fold) in a dose-dependent manner, while treatment with desvenlafaxine had no effect on drug efflux transporter expression. To determine the functional significance of this effect, the permeability of a known drug efflux probe, rhodamine 123, across the BBB model and Caco-2 cells, a model of intestinal absorption, were examined. Treatment with venlafaxine (1-50 µM) for 1 h significantly reduced the apical-to-basolateral permeability of R123 across the BBB model (30%) and Caco-2 cell monolayers (25%), indicative of increased drug efflux transporter expression at the apical membrane. Conversely, desvenlafaxine had no effect on R123 permeability in either cellular model. These studies indicate that venlafaxine, but not desvenlafaxine is an inducer of drug efflux transporter expression, which consequently increases the potential for clinical drug-drug interactions. Therefore, based on these preliminary results, caution should be taken when prescribing venlafaxine with other P-gp substrates.

Epitope-dependent effects of Beta-amyloid antibodies on Beta-amyloid clearance in an in vitro model of the blood-brain barrier.

OBJECTIVE: To investigate the role of RAGE in the epitope-dependent effects of Aß antibodies used as a peripheral sink therapy in AD. METHODS: An in vitro model of the BBB was used to examine the effect of various Aß antibodies or Aß peptide fragments on Aß exchange across the BBB. RESULTS: An N-terminal Aß antibody significantly enhanced the basolateral-to-apical transcytosis of fluorescein-Aß(1-42) across the BBB model (41%), while no effect was apparent with a C-terminal Aß antibody. Interestingly, modulation of RAGE in the presence of a C-terminal Aß antibody resulted in a 65% increase in Aß clearance across the BBB model, suggesting the C-terminal antibody-Aß complex is susceptible to RAGE transport. Additionally, N-terminal peptide fragments of Aß attenuated the brain penetration of full length Aß in the BBB model, indicating the N-terminal region of Aß is required for brain uptake. CONCLUSIONS: Antibodies masking the N-terminal region of Aß increase Aß clearance across the BBB by preventing Aß from interacting with the RAGE transporter, whereas antibodies bound to the C-terminus of Aß are taken up by RAGE and, hence, do not influence the BBB clearance of Aß.

Comparison of drug efflux transport kinetics in various blood-brain barrier models.

The present study quantitatively compared the drug efflux transport kinetics of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and its fluorescent metabolite 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in various blood-brain barrier (BBB) models. BCECF-AM was exposed to freshly isolated bovine brain microvessels (BBM), primary cultured bovine brain microvessel endothelial cells (BBMEC), and MDCK-MDR1 cells for 30 min in the presence or absence of the P-glycoprotein (P-gp) inhibitor N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918). P-gp transport kinetics were determined indirectly by calculating the difference in BCECF accumulation when P-gp was functional and completely inhibited by GF120918 (3.2 microM). Multidrug resistance-associated protein (MRP) transport kinetics were determined by measuring the amount of BCECF transported out of the cell over time. For P-gp-related transport, Km values for BCECF-AM were approximately the same in all three models (around 2 microM), whereas the Vmax was 4-fold greater in the BBM than in the BBMEC or MDCKII-MDR1 cells. For MRP-related transport, Km values for BCECF varied widely among the three BBB models with a rank order of MDCKII-MDR1 < BBMEC < BBM. Like P-gp, the Vmax of BCECF for MRP-related transport was overwhelmingly higher in the BBM compared with the cultured cells. Because differences in the expression of P-gp, MRP5, and MRP6 were observed in the various BBB models using reverse transcription-polymerase chain reaction techniques, the disparity in transport kinetics between the BBB models may be linked to variations in the amount or type of drug efflux transporters expressed in each model. The present study introduces a method of quantitatively evaluating drug efflux transport kinetics in the BBB.

Quantitative assessment of HIV-1 protease inhibitor interactions with drug efflux transporters in the blood-brain barrier.

PURPOSE: To quantitatively characterize the drug efflux interactions of various HIV-1 protease inhibitors in an in vitro model of the blood-brain barrier (BBB) and to compare that with HIV-1 protease inhibitor stimulated P-glycoprotein (P-gp)-ATPase activity. METHODS: Cellular accumulation of the P-gp sensitive probe, rhodamine 123 (R123), and the mixed P-gp/multidrug resistance-associated protein (MRP) probe, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), were evaluated in primary cultured bovine brain microvessel endothelial cells (BBMEC) in the presence of various concentrations of HIV-1 protease inhibitors. The potency (IC50) and efficacy (Imax) of the drugs in the cell accumulation assays for P-gp and/or MRP was determined and compared to activity in a P-gp ATPase assay. RESULTS: For R123 (P-gp probe), the rank order potency for inhibiting R123 accumulation in the BBMEC was saquinavir=nelfinavir>ritonavir=amprenavir>indinavir. This correlated well with the rank order affinity in the P-gp ATPase assay. The rank order potency for MRP-related drug efflux transporters, was nelfinavir>ritonavir>saquinavir>amprenavir>indinavir. CONCLUSIONS: HIV-1 protease inhibitors potently interact with both P-gp and MRP-related transporters in BBMEC. Characterization of the interactions between the HIV-1 protease inhibitors and drug efflux transporters in brain microvessel endothelial cells will provide insight into potential drug-drug interactions and permeability issues in the BBB.

A fluorometric screening assay for drug efflux transporter activity in the blood-brain barrier.

PURPOSE: To examine the capability of a fluorometric assay to identify and characterize the drug efflux interactions of a broad spectrum of drug agents in an in vitro model of the blood-brain barrier (BBB). METHODS: Various concentrations of drug agent (1, 10, and 100 microM) were evaluated for their effect on the cellular accumulation of the P-glycoprotein (P-gp) probe R123 (3.2 microM), and the mixed P-gp and multidrug resistance-associated protein (MRP) probe, BCECF (1 microM), in bovine brain microvessel endothelial cell (BBMEC) monolayers. Drugs demonstrating a significant effect were further quantitated using an expanded concentration range and a nonlinear regression curve fit to determine the potency (IC50) and efficacy (Imax) of the drug for P-gp and/or MRP. RESULTS: Several of the 36 therapeutic agents examined showed drug efflux transporter interactions in BBMEC monlayers. Melphalan and risperidone significantly enhanced the accumulation of R123 over control (1.47- and 1.82-fold, respectively) with resulting IC50s of 1.4 and 14.6 microM, respectively. Chlorambucil and valproic acid significantly enhanced the accumulation of BCECF compared to control monolayers (2.02- and 4.01-fold, respectively) with resulting IC50s of 146.1 and 768.5 microM, respectively. CONCLUSIONS: The current study demonstrates the feasibility of a fluorometric assay consisting of R123 and BCECF in assessing the drug efflux interactions of a variety of drugs in the BBB.

Drug efflux transport properties of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and its fluorescent free acid, BCECF.

2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) is a fluorescent probe used to examine multidrug resistance-associated protein (MRP) transporter activity in cells. BCECF is introduced into the cell as the nonfluorescent membrane permeable acetoxymethyl ester, BCECF-AM, where it is hydrolyzed to the membrane impermeable BCECF. The lipophilic nature of BCECF-AM suggests it may be a substrate for other drug efflux transporters such as P-glycoprotein (P-gp) and the breast cancer resistance protein (BCRP). To assess the drug efflux transporter interactions of BCECF-AM and BCECF, accumulation studies were examined in various drug efflux-expressing cells. Inhibition of P-gp, BCRP, and/or MRP produced distinct changes in the time-dependent accumulation of BCECF in the cells. Treatment with GF120918 produced an immediate and sustained effect throughout the entire time course examined. Fumitremorgin C only affected BCECF accumulation at the early time points, whereas the impact of indomethacin on BCECF accumulation was observed only at the latter time points. Permeability studies in bovine brain microvessel endothelial cells indicated an increased basolateral-to-apical transport of BCECF, which could be reduced in the presence of either indomethacin or GF120918. These results indicate that the intracellular accumulation and transcellular permeability of BCECF are sensitive to a variety of drug efflux interactions. These results likely reflect an interaction of the ester form with P-gp and BCRP during the initial accumulation process, and an interaction of the free acid form with MRP after hydrolysis in the cell.