Transport characteristics of zolmitriptan in a human intestinal epithelial cell line Caco-2

The intestinal absorption characteristics and the efflux mechanisms of zolmitriptan, a new generation and highly selective 5-HT(1B/1D) receptor agonist used in the acute oral treatment of migraine, were investigated. A human intestinal cell line, Caco-2, was used as an in-vitro model of the intestinal mucosa to assess transepithelial transport of zolmitriptan. In the Caco-2 cells, the absorptive transport of zolmitriptan was pH dependent and the transport was enhanced at weakly alkali pH on the apical side. No concentration dependence and saturation were observed for the apical-to-basolateral and basolateral-to-apical transport of zolmitriptan at a concentration of 0.1-10 mM. The permeability ratio value was about 1.5-2.6 at a concentration of 0.1-2.0 mM. Inhibition experiments using verapamil, nifedipine and nimodipine as inhibitors were studied and indicated that P-glycoprotein participated in the transport of zolmitriptan. Inhibition of the Na+-H+ exchanger with amiloride resulted in a significant increase in absorption and a slight inhibition in secretion. This suggests that the Na+-H+ exchanger may be involved in the transport of zolmitriptan. The results indicated that the transport of zolmitriptan was mediated by both passive diffusion and active transport. A series of drug-drug interaction experiments were carried out between zolmitriptan and some drugs that may be co-administrated with zolmitriptan in the clinic. The results indicated that flunarizine, cetirizine, propranolol and atenolol potently decreased both the apical-to-basolateral and basolateral-to-apical transport rate of zolmitriptan. Cimetidine and aspirin slightly inhibited the apical-to-basolateral transport of zolmitriptan, but significantly decreased the basolateral-to-apical transport of zolmitriptan. Thus, the absorption drug-drug interactions should be considered when these drugs are co-administrated with zolmitriptan in the clinic.     

Regulation of nitric oxide production in cultured human T84 intestinal epithelial cells by nuclear factor-kappa B-dependent induction of inducible nitric oxide synthase after exposure to bacterial endotoxin

BACKGROUND: Intestinal epithelium is consistently in contact with lipopolysaccharide (LPS) produced by intraluminal microbes. LPS induces nitric oxide production in many rodent cells, but in human cells it is very differently regulated. AIM: To test the hypothesis that exposure to LPS up-regulates nitric oxide synthesis in human intestinal epithelium. METHODS AND RESULTS: LPS induced nitric oxide synthesis in T84 cells in a time- and dose-dependent manner whereas detectable amounts of peroxynitrite were not produced. A novel selective inducible nitric oxide synthase (iNOS) inhibitor 1400 W potently inhibited LPS-induced nitric oxide synthesis in T84 cells while dexamethasone was relatively ineffective. Nitric oxide production was sensitive to cycloheximide, indicating that it was dependent on de novo protein synthesis. Nuclear factor-kappa B (NF-kappa B) inhibitor pyrrolidinedithiocarbamate abolished iNOS and nitric oxide production. Nitric oxide synthesis was also suppressed by genistein (tyrosine kinase inhibitor) and PD 98059 (p44/42 MAP kinase inhibitor) but enhanced by SB 203580 (p38 MAP kinase inhibitor). CONCLUSIONS: Intestinal epithelial cells express iNOS and produce nitric oxide in a nuclear factor-kappa B-dependent manner when exposed to LPS. The process is regulated by tyrosine kinases, and p44/42 and p38 MAP kinases. Because nitric oxide acts as an antimicrobial agent and immune modulator, these findings are implicated in the regulation of gut mucosal immunity.     

Effects of silkworm powder on glucose absorption by human intestinal epithelial cell line Caco-2

Inhibitors of glucose absorption play an important role in the treatment of diabetes. In this research, we report that silkworm powder has inhibitory effects on glucose absorption in human intestinal epithelial cells. Silkworm powder inhibited α-glucosidase activation and glucose transporter (SGLT1) expression. These results suggest that silkworm powder can be used as a natural functional food for the prevention and alleviation of type-2 diabetes.     

Transepithelial permeation of tolbutamide across the human intestinal cell line, Caco-2

Sulfonylurea hypoglycemic agents have interindividual variability in the gastrointestinal absorption rate. However, the absorption mechanism at the intestinal epithelium has not yet been clarified. To elucidate contribution of the specific mechanism for transepithelial transport of sulfonylureas, the apical-to-basolateral and basolateral-to-apical transport studies of tolbutamide were carried out using Caco-2 cell monolayers cultured on the polycarbonate membrane. The transported amounts of the substrate were measured by HPLC to estimate the apparent permeability coefficients (P(app)). In the apical-to-basolateral flux, the transport activity of tolbutamide was facilitated when the pH of the apical medium was more acidic than the basolateral one. ATP-depletion decreased the P(app) of tolbutamide. The kinetic analysis of the permeation rate indicated that the saturable process largely contributed to the tolbutamide flux. The P(app) of tolbutamide was lowered by an ionophore and monocarboxylic acids, while dicarboxylic acids and the inhibitor for the anion exchanger had no effect. In addition, mutual inhibition with benzoic acid was observed in transepithelial transport of tolbutamide. On the other hand, the permeation rate of tolbutamide from the basolateral to apical side was concentration-independent and neither affected by metabolic inhibitors, probenecid nor inhibitors for P-glycoprotein. In conclusion, these results suggest that apical-to-basolateral transport of tolbutamide across the Caco-2 cell monolayers is mediated by the pH-dependent specific system, presumably shared with other organic anions such as benzoic acid.     

Transport of levovirin prodrugs in the human intestinal Caco-2 cell line

The transport of 10 amino acid ester prodrugs of levovirin (LVV) was investigated in the human intestinal Caco-2 cell line in order to overcome the poor oral bioavailability of LVV, an investigational drug for the treatment of hepatitis C infection. The prodrugs were designed to improve the permeability of LVV across the intestinal epithelium by targeting the di/tri-peptide carrier, PepT1. Caco-2 cell monolayers were employed to study the transport and hydrolysis properties of the prodrugs. Among all mono amino acid ester prodrugs studied, the LVV-5'-(L)-valine prodrug (R1518) exhibited the maximum increase (48-fold) in permeability with nearly complete conversion to LVV within 1 h. Di-amino acid esters did not offer significant enhancement in permeability comparing with mono amino acid esters and exhibited slower conversion to LVV in Caco2 cell monolayers. Pharmacokinetic screening studies of the prodrugs in rats yielded the highest fold increase (6.9-fold) of AUC with R1518 and in general displayed a similar trend to that observed in increases of permeability in Caco-2 cells. Mechanisms involved in the Caco-2 cell transport of R1518 were also investigated. Results of bi-directional transport studies support the involvement of carrier-mediated transport mechanisms for R1518, but not for the LVV-5'-(D)-valine prodrug or LVV. Moreover, the permeability of R1518 was found to be proton dependent. PepT1-mediated transport of R1518 was supported by results of competitive transport studies of R1518 with the PepT1 substrates enalapril, Gly-Sar, valganciclovir, and cephalexin. R1518 was also found to inhibit the permeability of valganciclovir and cephalexin. These results suggest that R1518 is a PepT1 substrate as well as an inhibitor.     

Cytotoxicity of peptide-coated silver nanoparticles on the human intestinal cell line Caco-2

Silver nanoparticles are used in a wide range of consumer products such as clothing, cosmetics, household goods, articles of daily use and pesticides. Moreover, the use of a nanoscaled silver hydrosol has been requested in the European Union for even nutritional purposes. However, despite the wide applications of silver nanoparticles, there is a lack of information concerning their impact on human health. In order to investigate the effects of silver nanoparticles on human intestinal cells, we used the Caco-2 cell line and peptide-coated silver nanoparticles with defined colloidal, structural and interfacial properties. The particles display core diameter of 20 and 40 nm and were coated with the small peptide L-cysteine L-lysine L-lysine. Cell viability and proliferation were measured using Promegas CellTiter-Blue(?) Cell Viability assay, DAPI staining and impedance measurements. Apoptosis was determined by Annexin-V/7AAD staining and FACS analysis, membrane damage with Promegas LDH assay and reactive oxygen species by dichlorofluorescein assay. Exposure of proliferating Caco-2 cells to silver nanoparticle induced decreasing adherence capacity and cytotoxicity, whereby the formation of reactive oxygen species could be the mode of action. The effects were dependent on particle size (20, 40 nm), doses (5-100 μg/mL) and time of incubation (4-48 h). Apoptosis or membrane damage was not detected.     

Transport characteristics of grepafloxacin and levofloxacin in the human intestinal cell line Caco-2.

Transport characteristics of grepafloxacin and levofloxacin across the apical membrane of Caco-2 cells were examined. Both grepafloxacin and levofloxacin uptakes increased rapidly, and were temperature-dependent. Grepafloxacin and levofloxacin uptakes showed concentration-dependent saturation with Michaelis constants of 3.9 and 9.3 mM, respectively. Uptake of grepafloxacin and levofloxacin increased in Cl(-)-free and ATP depleted conditions, suggesting the involvement of an efflux transport system different from the uptake mechanism. However, cyclosporin A, a typical inhibitor of P-glycoprotein, did not affect the uptake of these drugs. Unlabeled grepafloxacin, unlabeled levofloxacin and quinidine inhibited the uptake of grepafloxacin and levofloxacin under Cl(-)-free conditions. Tetraethylammonium, cimetidine, p-aminohippurate, probenecid, amino acids, beta-lactam antibiotic or monocarboxylates did not inhibit the uptake of grepafloxacin and levofloxacin under the same conditions. In conclusion, our results suggested that grepafloxacin and levofloxacin uptakes were mediated by a specific transport system distinct from those for organic cations and anions, amino acids, dipeptides and monocarboxylates.     

As2O3-induced oxidative stress and cycle progression in a human intestinal epithelial cell line (Caco-2).

Foods and drinking water are the main routes for human exposure to inorganic arsenic, the intestinal epithelium being the first barrier against such exogenous toxicants. The present study evaluates the effect of As(III) (0.5-25 microM) upon Caco-2 cells as an intestinal epithelia model. Cell viability, intracellular formation of reactive oxygen species (ROS), mitochondrial membrane potential (Deltapsim) changes, and cell cycle distribution in exposed cultures were evaluated. The intracellular production of ROS was seen to increase in a non-dose dependent manner at all concentrations tested, with impairment of cell mitochondrial enzyme function secondary to a loss of Deltapsim. Concentrations between 0.5 and 5 microM induce cell cycle transition from phase G1 to phase S, with no significant alteration in the proportion of cells in phase G2. These data suggest that As(III) could induce intestinal oxidative stress-cytotoxicity at mitochondrial functional level, and affect cell cycle progression. The data presented in this work may also suggest the impairment of essential survival processes in Caco-2 cells, induced after exposure to As(III) (1-25 microM). Oxidative stress and alteration of mitochondrial functionality could be early indicators of arsenic-induced cytotoxicity, with the resulting abnormal progression of the cell cycle.     

Effects of progesterone and norethisterone on cephalexin uptake in the human intestinal cell line Caco-2

Little is known about the regulatory effects of steroid hormones on the intestinal H(+)/oligopeptide transporter PEPT1. In the present study, we investigated the effects of progesterone and its related compounds on the uptake of cephalexin, a typical PEPT1 substrate, using the human intestinal cell line Caco-2. Caco-2 cell monolayers were cultured on plastic cell culture plates coated with rat tail collagen type I. The determination of cephalexin uptake was performed with HPLC. To investigate the effect of progesterone treatment on cephalexin uptake, the monolayers were incubated with cephalexin after progesterone treatment. Progesterone decreased cephalexin uptake in a concentration-dependent manner, and the IC(50) value was calculated to be 2.3 microM. A significant decrease in cephalexin uptake was observed after progesterone treatment for 12 h, and the decrease was maximal when the monolayers were treated for 24-72 h. Cephalexin uptake was significantly inhibited by treatments with 17alpha-hydroxyprogesterone, norethisterone, and chlormadinone. By contrast, treatment with dehydroepiandrosterone, pregnenolone, estradiol, testosterone, hydrocortisone, and dexamethasone did not affect cephalexin uptake. The effects of progesterone and norethisterone treatment on the kinetic parameters of cephalexin uptake were investigated. The saturable component of cephalexin uptake was markedly inhibited by progesterone and norethisterone treatments. The J(max) of cephalexin uptake with progesterone and norethisterone treatments was significantly decreased compared with the control, whereas K(m) and K(d) values were not affected. Therefore the quantitative decrease in PEPT1 density is considered to be one cause of the decrease in cephalexin uptake with progesterone and norethisterone treatments.     

Modulation of the induction of nitric oxide synthase by eicosanoids in the murine macrophage cell line J774.

The effect of eicosanoids on the induction of nitric oxide synthase in the murine macrophage cell line J774 has been studied. We found that prostaglandin E2 (PGE2) and iloprost (a stable analogue of prostacyclin) both at nanomolar concentrations inhibited the lipopolysaccharide stimulated induction of NO synthase. In contrast PGF2 alpha, U46619, a stable analogue of thromboxane A2, leukotrienes B4 and C4 had no effect. These data demonstrate that the L-arginine: NO pathway in macrophages may be modulated by prostanoids.     

Transport of quercetin di-sodium salt in the human intestinal epithelial Caco-2 cell monolayer 139.

Quercetin di-sodium salt (QDS), a water-soluble derivative of quercetin (Q), is a potent free radical scavenger. The aim of this study was to examine the in vitro intestinal transport of QDS compared to that of Q using the Caco-2 human intestinal epithelial cell line. The apical (A) to basolateral (B) transport of QDS was found to be higher than the B to A transport of this compound. This polarized transport involved the presence of a carrier protein system. The involvement of the sodium/glucose transporter-1 (SGLT-1) was shown by using phloridzin, a selective inhibitor of this conveyor system. However, the transport of Q was not affected by this inhibitor. Moreover, the influx of QDS was pH-sensitive and decreased at pH 5.5 compared with that observed at pH 7.4 and 6.5. The permeability of QDS was 10-fold higher than that of Q. This could be explained by the involvement of SLGT-1 and the absence of an active efflux pump in the absorption of QDS in comparison with Q. This finding was supported by comparing the solubility of Q with that of QDS. This study indicates that both the higher solubility of QDS and its dependence on the SGLT-1 transport system resulted in more efficient permeability compared to Q.     

Isatins inhibit cyclooxygenase-2 and inducible nitric oxide synthase in a mouse macrophage cell line

Isatin is a versatile compound with a diversity of effects. We designed to investigate the inhibitory effect of isatin derivatives on lipopolysaccharide/interferon-gamma-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins, production of prostaglandin E(2) (PGE(2)), nitric oxide (NO), tumor necrosis factor (TNF-alpha), and their capacity to scavenge NO. Isatins inhibit TNF-alpha production and iNOS and COX-2 protein expression resulting on reduced levels of NO and PGE(2). Our results indicate isatin and it derivatives as inhibitors of iNOS and COX-2 enzymes, which might be used as anti-inflammatory and antitumoral agents.     

Active secretion of the fluoroquinolone ciprofloxacin by human intestinal epithelial Caco-2 cell layers.

The bidirectional transepithelial fluxes of ciprofloxacin, an antibacterial fluoroquinolone, across the human intestinal epithelial Caco-2 cell-line show marked asymmetry. Basal-to-apical flux of ciprofloxacin (10 microM) exceeds apical-to-basal flux indicating net secretion. Net ciprofloxacin secretion is abolished by azide/2-deoxy-D-glucose treatment, displays saturation kinetics (Km = 0.89 +/- 0.23 mM, Vmax 44.3 +/- 4.9 nmol cm-2.h) and competition by other fluoroquinolones. A specific, active secretion in Caco-2 epithelia may explain the transintestinal elimination of ciprofloxacin observed in pharmacokinetic studies in man.     

Transport characteristics of ebastine and its metabolites across human intestinal epithelial Caco-2 cell monolayers

The transport characteristics of a selective peripheral H1 receptor antagonist, ebastine, a substrate for cytochrome P450 3A4, and its three major metabolites, i.e., the hydroxy metabolite of ebastine (M-OH), the pharmacologically active metabolite carebastine (Car), and the desbutyrophenone metabolite (des-BP), were studied in cultured human intestinal Caco-2 cells expressing a drug efflux pump, P-glycoprotein (P-gp), on the apical membrane. The polarized transport of [3H]cyclosporin A (CyA), mediated by P-gp in the basolateral to apical direction across the Caco-2 cell monolayers, was affected by the presence of ebastine in a concentration-dependent manner and significant inhibition was observed at high concentrations (>50 microM). M-OH (300 microM) also significantly inhibited whereas Car and des-BP did not. Although no marked polarized transport of [14C]ebastine in a secretory direction was observed in the Caco-2 systems, the flux in the basolateral to apical direction was slightly higher than that in the opposite direction at concentrations less than 30 microm. [14C]Ebastine (2 microM) uptake from the apical side was significantly increased in the presence of an excess of cold CyA, suggesting that the efflux process mediated by P-gp may be involved in the ebastine uptake by Caco-2 cells. Collectively, these results indicate that ebastine (and presumably M-OH) is transported via P-gp in Caco-2 cells, however, the affinity for P-gp is very low. It is unlikely that the secretory transport of ebastine mediated by P-gp will dramatically affect overall intestinal absorption in vivo because efficient passive diffusion of this drug should occur due to its high lipophilicity. However, it may be advantageous for its efficient first-pass metabolism.     

Involvement of specific transport system on uptake of lactone form of SN-38 in human intestinal epithelial cell line Caco-2

The aim of this study was to elucidate the characteristics of the transport of lactone and carboxylate forms of SN-38 (SN-38L and SN-38C, respectively), a metabolite of irinotecan hydrochloride (CPT-11), with the human intestinal epithelial cell line, Caco-2. We examined SN-38L and SN-38C uptake from the apical side into Caco-2, and the effects of various compounds on the uptake of SN-38L. SN-38L and SN-38C in the cells were determined by HPLC with a fluorescence detector. When either SN-38L (0.5?μM) or SN-38C (0.5?μM) was added extracellularly at 37°C, the accumulation of SN-38L into the cells was about 10-fold higher than that of SN-38C, suggesting a dominant role of the lactone form in the uptake of SN-38 into Caco-2. The accumulation of SN-38L in Caco-2 increased time-dependently up to 10?min at 37°C, whereas the accumulation markedly decreased at 4°C. The initial uptake rate of SN-38L approached saturation at high concentrations with Michaelis-Menten constant and 'Hill coefficient,' 2.84±1.00?μM and 2.13±1.14, respectively (mean±S.E.). The accumulation of SN-38L was markedly inhibited by baicalin, an active ingredient of a Chinese herbal medicine, Hange-Shashin-To, as well as CPT-11. The type of inhibition by baicalin was competitive. In contrast, concomitant sulfobromophthalein, taurocholate and estrone 3-sulfate significantly increased SN-38L uptake. These results suggest that apical uptake of SN-38 by Caco-2 is dominantly performed as a lactone form through a specific transporter, which is competitively inhibited by baicalin.     

The induction of nitric oxide synthase and intestinal vascular permeability by endotoxin in the rat.

1. The effect of endotoxin (E. coli lipopolysaccharide) on the induction of nitric oxide synthase (NOS) and the changes in vascular permeability in the colon and jejunum over a 5 h period have been investigated in the rat. 2. Under resting conditions, a calcium-dependent constitutive NOS, determined by the conversion of radiolabelled L-arginine to citrulline, was detected in homogenates of both colonic and jejunal tissue. 3. Administration of endotoxin (3 mg kg-1, i.v.) led, after a 2 h lag period, to the appearance of calcium-independent NOS activity in the colon and jejunum ex vivo, characteristic of the inducible NOS enzyme. 4. Administration of endotoxin led to an increase in colonic and jejunal vascular permeability after a lag period of 3 h, determined by the leakage of radiolabelled albumin. 5. Pretreatment with dexamethasone (1 mg kg-1 s.c., 2 h prior to challenge) inhibited both the induction of NOS and the vascular leakage induced by endotoxin. 6. Administration of the NO synthase inhibitor NG-monomethyl-L-arginine (12.5-50 mg kg-1, s.c.) 3 h after endotoxin injection, dose-dependently reduced the subsequent increase in vascular permeability in jejunum and colon, an effect reversed by L-arginine (300 mg kg-1, s.c.). 7. These findings suggest that induction of NOS is associated with the vascular injury induced by endotoxin in the rat colon and jejunum.     

Transport and uptake characteristics of a new derivative of berberine(CPU-86017) by human intestinal epithelial cell line: Caco-2

AIM: The characteristics of transepithelial transport and uptake of CPU-86017 {[7-(4-chlorbenzyl)-7,8,13,13α-tetrahydroberberine chloride, CTHB]}, a new antiarrhythmia agent and a new derivative of berberine, were investigated on epithelial cell line (Caco-2) to further understand the absorption mechanism of berberine and its derivatives. METHODS: Caco-2 cell was used. RESULTS: 1) The permeability coefficient from the apical (AP) to basolateral (BL) of CPU-86017 was approximately 5 times higher than that from BL-to-AP transport. The effects of a P-glycoprotein (P-gp) inhibitor-cyclosporin A, some surfactants, and lower pH on the transepithelial transport of CPU-86017 were also observed. Cyclosporine A at 7.5 mg/L had no effect on the transepithelial electrical resistance (TEER); an about 4-fold enhancement on the transepithlial transport of CPU-86017 was observed. Some surfactants (sodium citrate, sodium deoxycholate, and sodium dodecyl sulfate) at 100 μmol/L and low pH (pH=6.0) induced a reversible d