Mechanism of L-α-Methyldopa Transport Through a Monolayer of Polarized Human Intestinal Epithelial Cells (Caco-2)

The Caco-2 model system (Hidalgo et al., Gastroenterology, 96:736–749, 1989), which is a monolayer of polarized intestinal epithelial cells grown onto a porous polycarbonate membrane, was used to study the mechanism of transcellular transport of an antihypertensive agent, L--methyldopa (L--MD). The results showed that the transport of L--MD was pH, glucose, concentration, and temperature dependent, and it could be inhibited by metabolic inhibitors (e.g., 2,4-dinitrophenol) and by amino acids (e.g., L-phenylalanine) which have an affinity for the large neutral amino acid (LNAA) carrier. In addition, the apparent kinetic constants describing the transcellular transport of L--MD were altered depending on the time interval between feeding the cells and the transport experiments (postfeeding time, PFT). The apparent maximum carrier flux (J _max) of L--MD was significantly increased (from 155 to 547 pmol/mg protein/min) when PFT was prolonged from 8.5 to 56 hr. These results indicated that the transcellular transport of L--MD through the polarized Caco-2 cell monolayer was carrier mediated via the LNAA carrier. The similarities in the characteristics of L--MD transport exhibited by the Caco-2 model system and other intestinal models in vitro further substantiate the usefulness of this cell culture model for studying the intestinal transport of nutrients and drugs.     

Are MDCK Cells Transfected with the Human MRP2 Gene a Good Model of the Human Intestinal Mucosa?

Purpose. To investigate whether Madin-Darby canine kidney cells transfected with the human MRP2 gene (MDCK-MRP2) are a good model of the human intestinal mucosa. Methods. MRP2 expression in Caco-2 cells was compared with the expression of this efflux transporter in MDCK-wild type (MDCK-WT) and MDCK-MRP2 cells using Western blotting methods. The polarized efflux activities of MRP2 in the MDCK-MRP2, MDCK-WT, MDCK cells transfected with the human MDR1 gene (MDCK-MDR1), and Caco-2 cells were compared using vinblastine as a substrate. Apparent Michaelis-Menten constants (K_M, Vmax) for the efflux of vinblastine in Caco-2 and MDCK-MRP2 cells were determined in the presence of GF120918 (2 M), which inhibits P-glycoprotein but does not affect MRP2. Apparent inhibitory constants (K_I) of known substrates/inhibitors of MRP2 were determined by measuring their effects on the efflux of vinblastine in these cell lines. Results. MDCK-MRP2 cells expressed higher levels of MRP2 than MDCK-WT and Caco-2 cells as measured by Western blotting technique. Two isoforms of MRP2 expressed in Caco-2 and MDCK cells migrated at molecular weights of 150 kD and 190 kD. In MDCK-MRP2 cells, the 150 kD isoform appeared to be overexpressed. MDCK-MRP2 cell monolayers exhibited higher polarized efflux of vinblastine than Caco-2 and MDCK-WT cell monolayers. K_M values for vinblastine in Caco-2 and MDCK-MRP2 cells were determined to be 71.8 ± 11.6 and 137.3 ± 33.6 M, respectively, and V_max values were determined to be 0.54 ± 0.03 and 2.45 ± 0.31 pmolcm^–2s^–1, respectively. Known substrates/inhibitors of MRP2 showed differences in their ability to inhibit vinblastine efflux in Caco-2 cells as compared to MDCK-MRP2 cells Conclusions. These data suggest that MDCK-MRP2 cells overexpress only the 150 kD isoform of MRP2. The 190 kD isoform, which was also found in Caco-2 cells and MDCK-WT cells, was present in MDCK-MRP2 cells but not over expressed. The apparent kinetics constants and affinities of some MRP2 substrates were different in Caco-2 cells and MDCK-MRP2 cells. These differences in substrate activity could result from differences in the relative expression levels of the MRP2 isoforms present in Caco-2 cells and MDCK-MRP2 cells and/or differences in the partitioning of substrates in these two cell membrane bilayers.     

Transport and Metabolism of the Tea Flavonoid (–)-Epicatechin by the Human Intestinal Cell Line Caco-2

Purpose: Tea flavonoids, including (–)-epicatechin (EC), have been suggested to have chemopreventive properties in cancer. However, there is limited knowledge of the oral bioavailability of these dietary compounds. The purpose of this study was to gain a better understanding of the absorption of EC. Methods: The intestinal epithelial membrane transport of EC was examined using the monolayer of the human Caco-2 cell line grown in Transwells, a common model of intestinal absorption. EC and its metabolites were measured by high performance liquid chromatography with diode array detection. Results: EC showed no apical to basolateral absorption at concentrations ranging from 5 to 50 M. In contrast, EC demonstrated basolateral to apical efflux with a P _app value of 0.67 ± 0.05 × 10^–6 cm/sec, i.e., slightly higher than for mannitol, 0.50 ± 0.30 × 10^–6 cm/sec, a paracellular transport marker. There was a 50% reduction in the efflux of EC in the presence of 50 M MK-571, a competitive inhibitor of the MRP2 transporter expressed in the apical membrane of Caco-2 cells. Most important, the presence of 50 M MK-571 resulted in clearly measurable apical to basolateral absorption of EC with a P _app of 0.31 ± 0.06 × 10^–6 cm/sec. Two polar metabolites, M1 and M2, were formed from EC, both of which appeared exclusively on the apical side. MK-571 (50 M) dramatically inhibited the transport for both of these metabolites. Incubations with inorganic ³⁵SO₄ ^2– and hydrolysis by aryl sulfatase strongly suggested that these metabolites were sulfate conjugates. Conclusions: These results suggest an important role for the multispecific organic anion transporter MRP2 in the bioavailability of EC and possibly other tea flavonoids.     

Inhibitory Effect of Zinc on PEPT1-Mediated Transport of Glycylsarcosine and β-Lactam Antibiotics in Human Intestinal Cell Line Caco-2

Purpose. The aim of this study was to examine the effects of zinc on the intestinal peptide transporters (PEPT1 and basolateral peptide transporter) and to elucidate the mechanism of the interactions. Methods. Caco-2 cells were pretreated with zinc, and the uptake studies were carried out. Results. Zinc treatment resulted in the inhibition of [¹⁴C]glycylsarcosine (Gly-Sar) uptake via PEPT1 in a concentration-dependent manner, whereas it showed moderate inhibitory effect on the basolateral peptide transporter. Zinc also inhibited the uptake of oral -lactam antibiotics such as ceftibuten and cephradine by PEPT1. Kinetic analysis showed that zinc treatment increased K _m values without affecting V _max values of the [¹⁴C]Gly-Sar uptake. The inhibition of [¹⁴C]Gly-Sar uptake induced by zinc was observed in the presence of an H⁺ gradient but not in the absence of an H⁺ gradient. Conclusions. These results indicate that zinc is a competitive inhibitor of PEPT1. Zinc inhibited the PEPT1 function, possibly by interacting with histidine residues of PEPT1 that are part of an H⁺-binding site. These findings would provide important information for clinical, physiologic, and biochemical aspects of peptide transporters.     

Are MDCK Cells Transfected with the Human MDR1 Gene a Good Model of the Human Intestinal Mucosa?

Purpose. To investigate whether Madin-Darby canine kidney cells transfected with the human MDR1 gene (MDCK-MDR1) are a good model of the human intestinal mucosa. Methods. P-glycoprotein (P-gp) expression in Caco-2 cells was compared with P-gp expression in MDCK wild- type (MDCK-WT) and MDCK-MDR1 cells using Western blotting methods. The polarized efflux activities of P-gp(s) in MDCK-MDR1 cells, MDCK-WT cells, and Caco-2 cells were compared using digoxin as a substrate. Apparent Michaelis-Menten constants (K _M,V _max) for the efflux of vinblastine in these three cell lines were determined. Apparent inhibition constants (K _I) of known substrates/inhibitors of P-gp were determined by measuring their effects on the efflux of digoxin in Caco-2 or MDCK-MDR1 cell monolayers. Results. MDCK-MDR1 cells expressed higher levels of P-gp compared to Caco-2 and MDCK-WT cells, as estimated by Western blots. Two isoforms of P-gp were expressed in Caco-2 and MDCK cells migrating with molecular weights of 150 kDa and 170 kDa. In MDCK-MDR1 cells, the 150 kDa isoforms appeared to be overexpressed. The MDCK-MDR1 cells exhibited higher polarized efflux of [³H]-digoxin than did Caco-2 and MDCK-WT cells. K _M values of vinblastine in Caco-2, MDCK-WT, and MDCK-MDR1 cells were 89.2 ± 26.1, 24.5 ± 1.1, and 252.8 ± 134.7 M, respectively, whereas V _max values were 1.77 ± 0.22, 0.42 ± 0.01, and 2.43 ± 0.86 pmolcm^–2s^–1, respectively. Known P-gp substrates/inhibitors showed, in general, lower K _I values for inhibition of digoxin efflux in Caco-2 cells than in MDCK-MDR1 cells. Conclusions. These data suggest that the MDCK-MDR1 cells overexpress the 150 kDa isoform of P-gp. MDCK-MDR1 cells are a useful model for screening the P-gp substrate activity of drugs and drug candidates. However, the apparent kinetics constants and affinities of substrates determined in the MDCK-MDR1 cell model may be different than the values obtained in Caco-2 cells. These differences in substrate activity could result from differences in the relative expression levels of total P-gp in Caco-2 and MDCK-MDR1 cells and/or differences in the partitioning of substrates into these two cell membrane bilayers.     

Changes in the NMR Metabolic Profile of Live Human Neuron-Like SH-SY5Y Cells Exposed to Interferon-α2

Interferon (IFN)-α2 is an extensively therapeutically used pro-inflammatory cytokine. Though its efficacy in controlling viral replication and tumor cells proliferation, administration of IFN-α2 is often associated with the development of central side effects. Magnetic resonance spectroscopy studies have demonstrated that IFN-α2 administration affects brain metabolism, however the exact nature of this effect is not completely known. We hypothesized that IFN-α2 can affect metabolic activity of human neuron-like SH-SY5Y cells which possess many characteristics of neurons and represent one of the most used models for studying mechanisms involved in neurotoxicity or neuroprotection. To test our hypothesis we have characterized the metabolic signature of live SH-SY5Y, and their conditioned media, after 24 and 72 h of exposure to vehicle or IFN-α2 (100 ng/ml) by using High Resolution-Magic Angle Spinning (HR-MAS) Nuclear Magnetic Resonance (NMR) spectroscopy. Our results revealed that 1) the use of HR-MAS NMR is ideally suitable for the characterization of the metabolic profile of live cells and their conditioned media without extraction procedures; and 2) a 72 h exposure to IFN-α2 increases the level of metabolites involved in maintaining energetic (including creatine and lactate) and osmotic (such as myo-inositol, scyllo-inositol, taurine and glycerophosphorylcholine) balances in neuron-like cells and of metabolic waste products (namely lactate, ethanol and acetate), glycine and glutamine in their growth media. These results may contribute to gain more knowledge about the IFN-α2 induced effect on the brain and support the interpretation of magnetic resonance spectroscopy studies performed in humans.     

Structural and Functional Differences Between Glycosylated and Non-glycosylated Forms of Human Interferon-β (IFN-β)

Purpose. Two recombinant IFN- products have been approved for the treatment of multiple sclerosis, a glycosylated form with the predicted natural amino acid sequence (IFN--la) and a non-glycosylated form that has a Met-1 deletion and a Cys-17 to Ser mutation (IFN--lb). The structural basis for activity differences between IFN--la and IFN--lb, is determined. Methods. In vitro antiviral, antiproliferative and immunomodulatory assays were used to directly compare the two IFN- products. Size exclusion chromatography (SEC), SDS-PAGE, thermal denaturation, and X-ray crystallography were used to examine structural differences. Results. IFN-- la was 10 times more active than IFN-- Ib with specific activities in a standard antiviral assay of 20 × 10⁷ lU/mg for IFN--la and 2 × 10⁷ lU/mg for IFN--lb. Of the known structural differences between IFN--la and IFN--lb, only glycosylation affected in vitro activity. Deglycosylation of IFN--la produced a decrease in total activity that was primarily caused by the formation of an insoluble disulfide-linked IFN precipitate. Deglycosylation also resulted in an increased sensitivity to thermal denaturation. SEC data for IFN--lb revealed large, soluble aggregates that had reduced antiviral activity (approximated at 0.7 × 10⁷ lU/mg). Crystallographic data for IFN--la revealed that the glycan formed H-bonds with the peptide backbone and shielded an uncharged surface from solvent exposure. Conclusions. Together these results suggest that the greater biological activity of IFN--la is due to a stabilizing effect of the carbohydrate on structure.     

The Effect of Benzyl Alcohol on Recombinant Human Interferon-γ

Purpose. The goal of this study was to investigate the conformational change and aggregation of recombinant human interferon-gamma (rhIFN-) as a result of interaction between benzyl alcohol and the protein. The effects of buffer concentration, buffer species, ionic strength, rhIFN- and benzyl alcohol concentrations on the dynamics of the interaction in liquid formulations were also examined. Methods. The effect of benzyl alcohol on the secondary and tertiary structure of rhIFN- in succinate and acetate buffers was studied using far-UV and near-UV circular dichroism spectrophotometry, respectively. Dynamic light scattering was employed to detect aggregate formation due to the interaction of benzyl alcohol with rhIFN-. Results. The addition of benzyl alcohol at 0.9% (w/v) in various liquid rhIFN- formulations induced changes in circular dichroism (CD) spectra of the protein in the near-UV region, while the CD spectra in the far-UV region remained unaltered. There were gradual decreases in ellipticity with time throughout the near-UV CD spectra. The decreases in near-UV ellipticity induced by benzyl alcohol were accompanied by the formation of high molecular weight aggregates as measured by dynamic light scattering. Loss in near-UV ellipticity was accelerated at lower protein concentration and by increasing buffer or benzyl alcohol concentration. It was also faster in succinate than in acetate buffer. Formulation ionic strength did not affect the CD spectral changes in both the near- and far-UV regions. Conclusions. Interaction between benzyl alcohol and rhIFN- is formulation dependent. Protein concentration, buffer species, buffer concentration, and preservative concentration play a significant role in determining the extent of the interaction and consequently the stability of the product.     

Genotoxicity Assessment of HM10620 Containing Recombinant Human Interferon-α

HM10620 is a recombinant human interferon-α (rhIFN-α) linked to immunoglobulin via N-terminal–specific non-peptidyl polyethylene glycol linker to improve the in vivo stability of interferon. Potential genotoxic effects of HM10620 in three short-term mutagenicity assays were investigated, which included the Ames assay, in vitro chromosomal aberration assay, and the in vivo micronucleus assay. HM10620 did not cause any mutation in the Ames assay tested using five tester strains at six concentrations of 6.25, 12.5, 25, 50, 100, and 200 μg/plate. To assess clastogenic effect, the in vitro chromosomal aberration assay and the in vivo micronucleus assay were performed using Chinese hamster lung cells and male ICR mice, respectively. Chromosomal aberration was not induced at the concentrations of 10, 20, and 40 μg/mL. Also, there was no difference in the incidence of micronucleated polychromatic erythrocytes at doses of 10, 20, and 40 mg/kg in male mice compared with the vehicle control group. Therefore, based on the results obtained from the three studies, it is concluded that HM10620 is not a mutagenic agent in bacterial cells and causes no chromosomal damage in mammalian cells both in vitro and in vivo.     

CriticalSorb? Promotes Permeation of Flux Markers Across Isolated Rat Intestinal Mucosae and Caco-2 Monolayers

Purpose

CriticalSorb? is a novel absorption enhancer based on Solutol^? HS15, one that has been found to enhance the nasal transport. It is in clinical trials for nasal delivery of human growth hormone. The hypothesis was that permeating enhancement effects of the Solutol^?HS15 component would translate to the intestine.

Methods

Rat colonic mucosae were mounted in Ussing chambers and Papp values of [¹⁴C]-mannitol, [¹⁴C]-antipyrine, FITC-dextran 4000 (FD-4), and TEER values were calculated in the presence of CriticalSorb?. Tissues were fixed for H &; E staining. Caco-2 monolayers were grown on Transwells? for similar experiments.

Results

CriticalSorb?(0.01%?v/v) significantly increased the Papp of [¹⁴C]-mannitol, FD-4 [¹⁴C]-antipyrine across ileal and colonic mucosae, accompanied by a decrease in TEER. In Caco-2 monolayers, it also increased the Papp of [¹⁴C]-mannitol FD-4 and [¹⁴C]-antipyrine over 120?min. In both monolayers and tissues, it acted as a moderately effective P-glycoprotein inhibitor. There was no evidence of cytotoxicity in Caco-2 at concentrations of 0.01% for up to 24?h and histology of tissues showed intact epithelia at 120?min.

Conclusions

Solutol^? HS15 is the key component in CriticalSorb? that enables non-cytotoxic in vitro intestinal permeation and its mechanism of action is a combination of increased paracellular and transcellular flux.     

Involvement of Transglutaminase-2 in α-MSH-Induced Melanogenesis in SK-MEL-2 Human Melanoma Cells

Skin hyperpigmentation is one of the most common skin disorders caused by abnormal melanogenesis. The mechanism and key factors at play are not fully understood. Previous reports have indicated that cystamine (CTM) inhibits melanin synthesis, though its molecular mechanism in melanogenesis remains unclear. In the present study, we investigated the effect of CTM on melanin production using ELISA reader and the expression of proteins involved in melanogenesis by Western blotting, and examined the involvement of transglutaminase-2 (Tgase-2) in SK-MEL-2 human melanoma cells by gene silencing. In the results, CTM dose-dependently suppressed melanin production and dendrite extension in α-MSH-induced melanogenesis of SK-MEL-2 human melanoma cells. CTM also suppressed α-MSH-induced chemotactic migration as well as the expressions of melanogenesis factors TRP-1, TRP-2 and MITF in α-MSH-treated SK-MEL-2 cells. Meanwhile, gene silencing of Tgase-2 suppressed dendrite extension and the expressions of TRP-1 and TRP-2 in α-MSH-treated SK-MEL-2 cells. Overall, these findings suggested that CTM suppresses α-MSH-induced melanogenesis via Tgase-2 inhibition and that therefore, Tgase-2 might be a new target in hyperpigmentation disorder therapy.     

The Effects of Pluronic® Block Copolymers and Cremophor® EL on Intestinal Lipoprotein Processing and the Potential Link with P-Glycoprotein in Caco-2 Cells

Purpose. This investigation was performed to study the effects of Pluronic® block copolymers and Cremophor® EL on intestinal lipoprotein processing and to investigate a potential link between lipoprotein processing and P-glycoprotein. Methods. Caco-2 cells were used to monitor changes in lipoprotein production and secretion following exposure to excipients. Effects on P-glycoprotein were monitored using cyclosporin A as a model substrate. Results. A range of surfactants commonly used as pharmaceutical excipients in lipid-based oral drug delivery systems, including Pluronic® block copolymers L81, P85, and F68 and Cremophor® EL, inhibited intestinal lipoprotein secretion. The effects were concentration dependent and reversible. The mechanism of inhibition appears to be related to the assembly and secretion of lipoproteins rather than to initial intracellular triglyceride synthesis. A strong correlation was found between excipient-mediated inhibition of lipoprotein secretion and inhibition of P-glycoprotein efflux, implying a link between the two biochemical processes. Conclusions. The ability of such bioactive excipients to simultaneously manipulate different cellular processes must be considered in selecting excipients for oral drug delivery systems. Such information is particularly relevant when the drug is lipophilic, a candidate for P-glycoprotein efflux, and where intestinal lymphatic targeting via chylomicron stimulation is desirable.     

A ‘Rule of Unity’ for Human Intestinal Absorption

The ability to predict the passive intestinal absorption of organic compounds can be a valuable tool in drug design. Although Lipinski's ‘rule of 5’ is commonly used for this purpose, it does not routinely give reliable results. An alternative ‘rule of unity’ is proposed to predict the absorption efficiency of orally administered drugs that are passively transported. The rule of unity based upon the theoretical principals that govern passive transport. The ‘rule of 5’ and the ‘rule of unity’ are compared using experimentally determined passive human intestinal absorption data for 155 drugs. Absorption values which are >50% of the dose are classified as well absorbed and absorption values which are ≤50% of the dose are classified as classified as poorly absorbed. Comparison of the two models using a receiver operating characteristic (ROC) plot and McNemar's test reveal striking differences in absorption predictability. The ‘rule of 5’ gives twice as many false predictions than the ‘rule of unity.’     

Selective Expression of CYP2A13 in Human Pancreatic α-Islet Cells

Exposure to cigarette smoke is an etiological factor of human pancreatic cancer and has been associated with an increased risk of pancreatic diseases, including pancreatitis and diabetes. The toxicants in cigarette smoke can reach pancreatic tissue, and most of the toxicants require cytochrome P450 (P450)-mediated metabolic activation to exert their toxicity. Among all the human P450 enzymes, CYP2A13 is the most efficient enzyme in the metabolic activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a major tobacco-specific toxicant and a suspected human carcinogen. It also metabolically activates 4-aminobiphenyl, another toxicant in cigarette smoke. Immunohistochemical analysis in this study demonstrated that CYP2A13 was selectively expressed in the islets but not in the exocrine portion of adult human pancreas. Further study using dual immunofluorescence labeling technique showed that CYP2A13 protein was mainly expressed in the α-islet but not in β-islet cells. The selective expression of CYP2A13 in human pancreatic α-islet cells suggests that these islet cells could be damaged by the toxicants existing in cigarette smoke through CYP2A13-mediated in situ metabolic activation. Our result provides a mechanistic insight for human pancreatic diseases that have been associated with cigarette smoke exposure.     

Use of the InteliSite® Capsule to Study Ranitidine Absorption from Various Sites Within the Human Intestinal Tract

Purpose. The purpose of this study was to evaluate the extent of ranitidine absorption from an externally activated drug-delivery system in two distinct regions of the intestine (jejunum and ileum) in healthy human volunteers. This investigation also was designed to evaluate the utility of the InteliSite ^® capsule for studying regional intestinal drug absorption in humans. Methods. The intestinal absorption of ranitidine from the jejunum and ileum was compared in eight, healthy volunteers in this open-label, two-way crossover study. In two of the eight volunteers, absorption from the colon also was studied. Subjects swallowed the capsule containing ranitidine solution (121 mg) and 100 Ci of ^99mTc-DTPA. The endcap of the capsule contained 20 Ci of ¹¹¹In-DTPA. At the desired intestinal site, the capsule was activated by the application of an external RF magnetic signal (6.78 MHz operating frequency) and the ranitidine solution was released. Blood samples were collected from a forearm vein for 12 hours after capsule activation. Results. The capsule released the ranitidine solution when activated in the jejunum, ileum and colon (visualized by the gamma camera). There was no difference in the extent of ranitidine absorption or ranitidine pharmacokinetics when the capsule was activated in the jejunum or ileum. Conclusions. This study demonstrates the utility of a novel, externally activated drug-delivery system to assess site-specific intestinal drug absorption in humans. Results indicate that use of the InteliSite ^® capsule method to evaluate site-specific intestinal ranitidine absorption in humans yields data similar to that obtained previously by means of oral intubation studies.     

Uptake and Transport of Novel Amphiphilic Polyelectrolyte-Insulin Nanocomplexes by Caco-2 Cells—Towards Oral Insulin

Purpose  

The influence of polymer architecture on cellular uptake and transport across Caco-2 cells of novel amphiphilic polyelectrolyte-insulin nanocomplexes was investigated.     

The Changes of P-glycoprotein Activity by Interferon-γ and Tumor Necrosis Factor-α in Primary and Immortalized Human Brain Microvascular Endothelial Cells

The purpose of this study was to investigate the modification of expression and functionality of the drug transporter P-glycoprotein (P-gp) by tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) at the blood-brain barrier (BBB). We used immortalized human brain microvessel endothelial cells (iHBMEC) and primary human brain microvessel endothelial cells (pHBMEC) as in vitro BBB model. To investigate the change of p-gp expression, we carried out real time PCR analysis and Western blotting. To test the change of p-gp activity, we performed rhodamin123 (Rh123) accumulation study in the cells. In results of real time PCR analysis, the P-gp mRNA expression was increased by TNF-α or IFN-γ treatment for 24 hr in both cell types. However, 48 hr treatment of TNF-α or IFN-γ did not affect P-gp mRNA expression. In addition, co-treatment of TNF-α and IFN-γ markedly increased the P-gp mRNA expression in both cells. TNF-α or IFN-γ did not influence P-gp protein expression whatever the concentration of cytokines or duration of treatment in both cells. However, P-gp expression was increased after treatments of both cytokines together in iHBMEC cells only compared with untreated control. Furthermore, in both cell lines, TNF-α or IFN-γ induced significant decrease of P-gp activity for 24 hr treatment. And, both cytokines combination treatment also decreased significantly P-gp activity. These results suggest that P-gp expression and function at the BBB is modulated by TNF-α or/and IFN-γ. Therefore, the distribution of P-gp depending drugs in the central nervous system can be modulated by neurological inflammatory diseases.