Change in tolbutamide permeability in rat jejunum and Caco‐2 cells by Sho‐saiko‐to (Xiao Chai Hu Tang), a Chinese traditional medicine

Objectives This study was designed to investigate the effects of Sho‐saiko‐to (Xiao Chai Hu Tang), a Chinese traditional medicine, on the membrane permeability of tolbutamide in the intestinal tract. We carried out an in‐situ loop study with rat jejunum and a transport study with Caco‐2 cell monolayers. Methods In the in‐situ loop study, absorption clearance of tolbutamide was estimated from the drug concentrations in the loop and plasma. The apical‐to‐basolateral and basolateral‐to‐apical transport of tolbutamide and d ‐mannitol, a paracellular transport marker, was assessed using Caco‐2 cell monolayers cultured on a polycarbonate membrane. Key findings The absorption clearance of tolbutamide was enhanced by a concomitant dose of Sho‐saiko‐to over 10 min in the rat in‐situ loop. Sho‐saiko‐to increased the apical‐to‐basolateral transport of tolbutamide, whereas the basolateral‐to‐apical transport of this drug was reduced by Sho‐saiko‐to. On the other hand, in both directions the P_app of d ‐mannitol was reduced by the presence of Sho‐saiko‐to. Furthermore, the apical‐to‐basolateral transport of tolbutamide in ATP‐depleted Caco‐2 cells was diminished by Sho‐saiko‐to. These findings suggest that Sho‐saiko‐to can facilitate the epithelial membrane permeability of tolbutamide across the rat jejunum in‐situ and Caco‐2 cell monolayers. Since Sho‐saiko‐to suppressed the passive transport of tolbutamide from the apical‐to‐basolateral side, enhanced permeability may be related to effects of Sho‐saiko‐to on the energy‐dependent transport of tolbutamide in the intestine. Conclusions Our findings suggest that Sho‐saiko‐to might facilitate the energy‐dependent transport of tolbutamide across the rat jejunum in‐situ and Caco‐2 cell monolayers.     

Distribution characteristics of clarithromycin and azithromycin, macrolide antimicrobial agents used for treatment of respiratory infections, in lung epithelial lining fluid and alveolar macrophages

The distribution characteristics of clarithromycin (CAM) and azithromycin (AZM), macrolide antimicrobial agents, in lung epithelial lining fluid (ELF) and alveolar macrophages (AMs) were evaluated. In the in vivo animal experiments, the time‐courses of the concentrations of CAM and AZM in ELF and AMs following oral administration (50 mg/kg) to rats were markedly higher than those in plasma, and the area under the drug concentration–time curve (AUC) ratios of ELF/plasma of CAM and AZM were 12 and 2.2, and the AUC ratios of AMs/ELF were 37 and 291, respectively. In the in vitro transport experiments, the basolateral‐to‐apical transport of CAM and AZM through model lung epithelial cell (Calu‐3) monolayers were greater than the apical‐to‐basolateral transport. MDR1 substrates reduced the basolateral‐to‐apical transport of CAM and AZM. In the in vitro uptake experiments, the intracellular concentrations of CAM and AZM in cultured AMs (NR8383) were greater than the extracellular concentrations. The uptake of CAM and AZM by NR8383 was inhibited by ATP depletors. These data suggest that the high distribution of CAM and AZM to AMs is due to the sustained distribution to ELF via MDR1 as well as the high uptake by the AMs themselves via active transport mechanisms. Copyright © 2011 John Wiley & Sons, Ltd.     

Transport characteristics of candesartan in human intestinal Caco‐2 cell line

The intestinal absorptive characteristics and the efflux mechanisms of candesartan (CDS), a novel angiotensin II type 1 receptor blocker, were investigated. The Caco‐2 cells were used as models of the intestinal mucosa to assess uptake and transport of CDS. The determination of CDS was performed by HPLC‐Flu. In the Caco‐2 cells, the uptake and absorptive transport of CDS were pH‐independent (in the pH range 6.0–8.0). Passive membrane diffusion dominates the absorptive transport behavior of CDS across Caco‐2 cells, while secretory transport was a concentration‐dependent and saturable process. In the presence of cyclosporin A and verapamil, potent inhibitors of P‐glycoprotein (P‐gp), the P_ratio decreased from 3.8 to 2.3 and 1.8, respectively, and permeation of apical to basolateral was enhanced. Overall, the current study suggests that efflux transporters are capable of mediating the absorption and secretion of CDS, and they may play significant roles in limiting the oral absorption of CDS. Copyright © 2009 John Wiley & Sons, Ltd.     

Role of ABC transporters in trans‐epithelial transport of vitamin K antagonists

Vitamin K antagonists (VKAs) remain the oral anticoagulant of choice in venous thromboembolic disease. These drugs are characterized by a large inter‐individual variability requiring frequent dose tailoring. Genetic polymorphisms for cytochrome CYP2C9 and VKORC1 explain some of the variability, especially in warfarin and acenocoumarol responses. The aim of this study was to assess, in cell models, the role of ABC transporters in the intestinal transfer of the main coumarin derivatives (warfarin, acenocoumarol) and indanedione derivatives (phenindione, fluindione). The results show a basal to apical polarized transport for fluindione, phenindione and acenocoumarol only. Experimental studies using specific inhibitors of transport protein demonstrate the implication of MRPs and BCRP proteins and to a lesser extent P‐gp. Warfarin and acenocoumarol seem to be poor inhibitors of MRPs protein, whereas fluindione and phenindione have a slight or no effect. The regulation of the expression of ABC transporters by exposure to VKAs was also investigated in Caco‐2 cells. The expression of mRNA P‐gp, MRP1, MRP2 and BCRP was weakly or not modified after 24 h of VKAs exposure. In conclusion, the intestinal transfer of indanedione derivatives and acenocoumarol could be influenced by transport proteins of the ABC superfamily. Coumarin derivatives are poor inhibitors of these proteins and AVKs have a slight effect on the mRNA ABC transporter expression level. Copyright © 2016 John Wiley & Sons, Ltd.     

Quercetin and naringenin transport across human intestinal Caco‐2 cells

Objectives Flavonoids are phenolic compounds found in most edible fruits and vegetables. Previous studies have demonstrated their biological and beneficial effects on human health. However, their bioavailability and, in particular, their intestinal absorption mechanism have not yet been clearly identified. The aim of our work was to quantify and to characterize in vitro the nature of the transport of two flavonoids distinguished by their physicochemical and pharmacological properties: quercetin, a flavan‐3‐ol, and naringenin, a flavanone. Methods Differentiated and polarized Caco‐2 human intestinal epithelial cell lines were used for this purpose. Key findings In our experimental conditions, quercetin and naringenin were poorly absorbed by Caco‐2 cells. Quercetin was absorbed by passive diffusion and a pH‐dependent mechanism mediated by the organic anion transporting protein B (OATP‐B). It was not a multidrug resistance associated protein (MRP)1 substrate, but was substrate of the MRP2 efflux transporter and not P‐glycoprotein (P‐gp). Intestinal permeability from the apical to the basolateral side was higher for naringenin than for quercetin, which was partly explained by naringenin's physicochemical characteristics. Naringenin, partially absorbed by passive diffusion, was also an ATP‐dependent transport substrate mediated by MRP1, but was not an OATP‐B substrate. However, naringenin was secreted via active P‐gp and MRP2 efflux transporters. Conclusions The contribution of ATP‐dependent efflux transporters (MRP2 and P‐gp) to the permeability of these compounds in the apical side could explain their low bioavailability. In conclusion, knowledge of the absorption mechanism of these two flavonoids was used to determine the intake level that has a beneficial effect on human health and their putative role in food—drug interactions.     

The membrane transport of flavonoids from Crossostephium chinense across the Caco-2 monolayer

The permeation and efflux of six polysubstituted flavonoids isolated from Crossostephium chinense, a Chinese traditional and herbal drug for the treatment of diabetes, were investigated using the Caco‐2 cell monolayer. The six flavonoids (selagin, apometzgerin, tricetin‐3′,4′,5′‐trimethylether, quercetagetin‐3,6,7‐trimethylether, hispidulin and quercetagetin) sharing a similar parent skeleton structure with varied substituents in the heterocyclic ring B were selected for the study. Quercetagetin exhibited a low bi‐directional permeability comparable to that of atenolol, suggesting a paracellular diffusion mechanism. The remaining compounds exhibited time‐ and concentration‐ dependent permeation with apparent permeability coefficient (P_app) values in the range 10^?6–10^?5 cm/s, suggesting transcellular diffusion pathways. Selagin exhibited significantly larger basolateral to apical P_app than that of the reverse direction, suggesting the existence of efflux mechanisms. ATP‐depletion and probenecid pretreatment led to a significant inhibition of the efflux of selagin, whereas verapamil had no effect on the basolateral to apical transport of selagin, suggesting that multidrug resistance proteins (MRPs) and not P‐glycoprotein play a role in the intestinal efflux of selagin. Based on present and previous results, the structure–permeation relationship and the role of MRPs in mediating the efflux of flavonoids are discussed. Experimental results in this study provide useful information for pharmacological applications of the flavonoids from C. chinense. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of efflux transport of the PDE5 inhibitors, vardenafil and sildenafil

Objectives We aimed to characterize the efflux transport properties of vardenafil and sildenafil, and to compare the kinetics of these compounds via efflux transporters such as P‐gp, BCRP and MRP2. Methods We measured the basal‐to‐apical and apical‐to‐basal transport of vardenafil and sildenafil within the concentration range of 1–100 µm using MDCKII cells overexpressing P‐gp, BCRP and MRP2, and Caco‐2 cells. Key findings Vardenafil had a much greater basal‐to‐apical than apical‐to‐basal transport rate in MDCKII cells overexpressing P‐gp, BCRP and MRP2. Sildenafil showed P‐gp‐ and BCRP‐mediated efflux transport, but did not seem to be pumped out via MRP2 transporters. Consequently, the absorptive transport of vardenafil and sildenafil in Caco‐2 cells increased linearly over the concentration range of 1–100 µm , whereas the secretory transport of these drugs was saturable and inhibited by the presence of specific inhibitors of P‐gp and BCRP. MK571, a representative MRP2 inhibitor, inhibited the basal‐to‐apical transport of vardenafil, but not of sildenafil. Conclusion The involvement of P‐gp, BCRP and MRP2 for vardenafil and the involvement of P‐gp and BCRP for sildenafil in the secretory transport with linear absorptive transport may contribute to the limited intestinal absorption of these drugs.     

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. Copyright © 2011 John Wiley & Sons, Ltd.     

Monoketocholate can decrease transcellular permeation of methotrexate across Caco‐2 cell monolayers and reduce its intestinal absorption in rat

Objectives Bile salts have been shown to decrease the absorption of methotrexate in the rat intestine by an unknown mechanism. We aimed to examine this effect. Methods We assessed apical‐to‐basolateral (AP‐BL) permeation of methotrexate (5 μM) across Caco‐2 cell monolayers pretreated with various concentrations (0, 0.25, 0.5, 1, 3 and 5 mm ) of sodium cholate or its semisynthetic analogue, sodium 12‐monoketocholate. We also determined the effect of orally administered 12‐monoketocholate on the intestinal absorption of methotrexate in rats to evaluate a possible in‐vitro–in‐vivo correlation. Key findings It was found that sodium cholate and sodium 12‐monoketocholate decreased the AP‐BL permeation of methotrexate at low concentrations (maximal inhibition at 0.25 and 1 mm , respectively) and increased it at higher concentrations. Determination of [¹⁴C] mannitol permeation and electrical resistance of monolayers during experiments showed that membrane integrity was not compromised at low concentrations of bile salts but was disrupted at higher concentrations. Subsequently, we examined the effect of the simultaneous oral administration of sodium 12‐monoketocholate (4, 20, 40 and 80 mg/kg) on the intestinal absorption of methotrexate in rats after an oral dose (5 mg/kg). The pharmacokinetic study showed that 12‐monoketocholate at 4 and 20 mg/kg did not change the methotrexate area under the serum concentration–time curve whereas sodium 12‐monoketocholate at 40 and 80 mg/kg significantly reduced it. Conclusions Sodium 12‐monoketocholate appears to decrease the intestinal absorption of methotrexate in rats by inhibition of transcellular active transport.     

Contribution of equilibrative nucleoside transporter(s) to intestinal basolateral and apical transports of anticancer trifluridine

Trifluridine (FTD) exhibits anticancer activities after its oral administration despite its hydrophilic nature. It was previously reported that concentrative nucleoside transporter (CNT) 1 mediates the apical uptake of FTD in human small intestinal epithelial cells (HIECs). In the present study, FTD was also identified as a substrate for equilibrative nucleoside transporter (ENT) 1 and ENT2 in transporter gene‐transfected cells. An immunocytochemical analysis revealed that ENT1 was expressed at the basolateral and apical membranes of HIECs. Cellular accumulation increased in the presence of S‐(4‐nitrobenzyl)‐6‐thioinosine (NBMPR), an ENT selective inhibitor. Cytotoxicity in HIEC monolayers at low FTD concentrations was increased by NBMPR, and this may have been due to inhibition of the ENT‐mediated basolateral transport of FTD by NBMPR. These results suggest that ENTs reduce the intestinal cytotoxicity of FTD by facilitating its basolateral efflux. On the other hand, the intracellular accumulation and cytotoxicity of FTD in HIECs were decreased at higher concentrations of FTD by NBMPR, and this may have been due to the NBMPR inhibition of the apical uptake of FTD, which has been suggested to be mediated by CNTs and ENTs. In conclusion, ENTs were responsible for intestinal transepithelial permeation by mediating the basolateral efflux of FTD after its uptake by CNT1 from the apical side, resulting in decreases in its intracellular accumulation and intestinal toxicity in humans. Equilibrative nucleoside transporters may also partially contribute to the low‐affinity uptake of FTD across the apical membrane along with high‐affinity CNT1.     

Intestinal transport of bis(12)-hupyridone in Caco-2 cells and its improved permeability by the surfactant Brij-35

The objective of the present study was to elucidate the mechanisms of intestinal transport of bis(12)‐hupyridone (B12H) to predict its oral bioavailability. The effect of the B12H concentration and the contribution of the drug efflux transporters, P‐glycoprotein (P‐gp or ABCB1) and multidrug resistance‐associated proteins (MRPs or ABCC) on B12H absorption were measured and evaluated using the human intestinal epithelial Caco‐2 cell monolayer in the presence of transporter inhibitors. The results indicated that B12H was absorbed in a dose‐dependent manner at concentrations ranging from 132 to 264 µM . However, only apical efflux was observed in the directional transport studies for B12H below 88 µM (P_app(AP‐to‐BL): virtually zero; P_app(BL‐to‐AP): 1.591 ± 0.071 × 10^?5 cm s^?1). P‐gp and mixed P‐gp/MRP inhibitors significantly increased the absorptive transport (P_app(AP‐to‐BL)) to 0.619 ± 0.018 × 10^?5 and 0.608 ± 0.025 × 10^?5 cm s^?1, respectively, while decreasing secretory transport (P_app(BL‐to‐AP)) by >75%. A multiple‐MRP inhibitor, probenecid, increased the P_app(AP‐to‐BL) to 0.329 ± 0.015 × 10^?5 cm s^?1 while decreasing the P_app(BL‐to‐AP) by 50%. Another multiple‐MRP inhibitor, indomethacin, only modestly decreased the P_app(BL‐to‐AP) by ～30% and had no effect on the absorptive transport (P_app(AP‐to‐BL): virtually zero). In addition, the effect of various pharmaceutical excipients (e.g. Pluronic F‐68, Tween‐80 and Brij‐35) on B12H transport was determined and compared. Among them, Brij‐35 effectively enhanced B12H absorption at a concentration lower than its critical micelle concentration (CMC, 60 µM ). Therefore, Brij‐35 can be used as a potential enhancer to improve intestinal absorption of B12H for oral administration. Copyright © 2011 John Wiley & Sons, Ltd.     

Enhancement by adrenaline of ginsenoside Rg1 transport in Caco‐2 cells and oral absorption in rats

Objectives The purpose of this research was to evaluate the ability of adrenaline (epinephrine) to stimulate the uptake of ginsenoside Rg1 (Rg1) by Caco‐2 cells. Methods Rg1 uptake was measured using Caco‐2 cell monolayers. The Rg1 uptake medium with adrenaline at different concentrations was added to each well and incubated for different time intervals. Adrenergic antagonists such as phentolamine and propranolol were added to the incubation medium to investigate their effects on Rg1 uptake. The Rg1 concentration in the monolayers was determined by high‐performance liquid chromato‐graphy. Transport of Rg1 across Caco‐2 cells was also studied and an oral bioavailability study of Rg1 was carried out in rats. Key findings The incubation medium with adrenaline remarkably increased the amount of Rg1 uptake by Caco‐2 cells. Adrenaline‐induced Rg1 transport increased in a dose‐ and time‐dependent manner. The effect of adrenergic antagonists on adrenaline‐induced uptake of Rg1 was investigated and it was found that the enhancement effect was attenuated by the co‐treatment with propranolol but not phentolamine. The transport amount of Rg1 by Caco‐2 cells increased in response to 1 mM adrenaline, isoproterenol or salbutamol. In contrast, 1 mM phenylephrine had no effect on Rg1 transport in Caco‐2 cells. The effect of adrenaline on the absorption of Rg1 was further investigated in vivo in rats. The co‐administration with adrenaline in rats showed that the oral bioavailability was increased remarkably relative to the aqueous solution. The area under the plasma concentration‐time curve of Rg1 after co‐administration with 1 mM adrenaline was 79.1 ± 31.04 μg/ml/h compared with 2.81 ± 1.13 μg/ml/h for its aqueous solution. Conclusions Adrenaline is effective for the stimulation of intestinal absorption of Rg1 and the enhanced absorption is mediated mainly by the interaction of adrenaline with β₂‐adrenoceptors.     

Enhancing effect of N-octyl-O-sulfate chitosan on etoposide absorption

P-glycoprotein (P-gp), expressed in the apical membranes of the epithelial cells of the intestine, can reduce the oral bioavailability of a wide range of drugs. Many surfactants/excipients have been demonstrated to potentially increase drug absorption by inhibiting P-gp. The purpose of the present study was to evaluate the effect of N-octyl-O-sulfate chitosan (NOSC) on the absorption of etoposide (VP16), a substrate of P-gp with low water solubility. The rat intestinal circulating perfusion in situ and Caco-2 cell uptake and monolayer membrane penetration in vitro were performed to investigate the enhancing ability of NOSC in comparison with some other P-gp inhibitors. The results indicated that various concentrations of NOSC all increased the intestinal absorption of VP16 in rat jejunum and ileum obviously and there was no significant difference in ileum between the enhancing effects of NOSC and other P-gp inhibitors. The VP16 uptake of Caco-2 cell was increased by NOSC solution with different concentrations. As the NOSC concentration was close to its critical micelle concentration (CMC), the cell uptake of VP16 reached to a maximum value. Both NOSC and verapamil (Ver) enhanced dramatically the transport of VP16 from apical side to basolateral side in Caco-2 cell monolayers. Moreover, they both decreased notably the transport of VP16 from basolateral side to apical side, but this effect of NOSC was weaker than that of Ver. However, transepithelial electrical resistance (TEER) of Caco-2 cell monolayers had no significant change during the study. These studies demonstrated that NOSC had the potential by inhibiting P-gp to improve the absorption of oral drugs which were P-gp substrates.     

Effect of Iron Treatment on Nickel Absorption and Gene Expression of the Divalent Metal Transporter (DMT1) by Human Intestinal Caco‐2 Cells

Abstract: Divalent Metal Transporter 1 (DMT1) is a transmembrane transporter located at the apical membrane of enterocytes and implicated in the duodenal uptake of iron. Results from expression experiments in Xenopus oocytes indicate that DMT1 can mediate transport of a wide range of divalent metals other than iron. The aim of the present study was to examine the effect of iron treatment on the uptake and transepithelial movement of ⁶³Ni and to correlate that to DMT1 messenger RNA (mRNA) levels in human intestinal Caco‐2 cells. Twenty‐one days after confluent Caco‐2 cell monolayers were treated for 1 or 3 days with medium supplemented with Fe(NTA)₂ or control medium and ⁶³Ni transport and DMT1 gene expression were measured at both time points. Functional effects of the iron treatment were assessed by examining uptake and transepithelial movement of ⁵⁹Fe. Iron treatment resulted in decreased DMT1 gene expression which correlated well with the uptake of ⁵⁹Fe and ⁶³Ni into fully differentiated Caco‐2 cells. This indicates that DMT1 is responsible for the apical transport of these metals in the intestinal epithelium and suggests that adequate iron intake and status will limit nickel absorption.     

Effects of Pluronic P85 Unimers and Micelles on Drug Permeability in Polarized BBMEC and Caco-2 Cells

Purpose. Using polarized bovine brain microvessel endothelial cells (BBMEC) monolayers as in vitro model of the blood brain barrier and Caco-2 monolayers as a model of the intestinal epithelium, the present work investigates the effects of Pluronic P85 block copolymer (P85) on the transport of the P-gycoprotein (P-gp)- dependent probe, rhodamine 123 (R123). Methods. The permeability and cell efflux studies are performed with the confluent cell monolayers using Side-Bi-Side diffusion cells. Results. At concentrations below the critical micelle concentration, P85 inhibits P-gp efflux systems of the BBMEC and Caco-2 cell monolayers resulting in an increase in the apical to basolateral permeability of R123. In contrast, at high concentrations of P85 the drug incorporates into the micelles, enters the cells and is then recycled back out to the apical side resulting in decrease in Rl 23 transport across the cell monolayers. Apical to basolateral permeability of micelle-incorporated R123 in BBMEC monolayers was increased by prior conjugation of P85 with insulin, suggesting that modified micelles undergo receptor-mediated transcytosis. Conclusions. Pluronic block copolymers can increase membrane transport and transcellular permeability in brain microvessel endothelial cells and intestinal epithelium cells. This suggests that these block copolymers may be useful in designing formulations to increase brain and oral absorption of select drugs.     

Role of basolateral efflux transporter MRP4 in the intestinal absorption of the antiviral drug adefovir dipivoxil

Adefovir dipivoxil is a diester prodrug of the antiviral drug adefovir, with much greater oral bioavailability than adefovir. Evidence shows that the prodrug is metabolized to adefovir in the enterocytes during intestinal absorption. However, it is unknown how the highly charged and hydrophilic adefovir crosses the basolateral membrane in the intestine. This study determines the role of specific basolateral transporter(s) in the egress of adefovir across the basolateral membrane when formed from adefovir dipivoxil in Caco-2 cells, a model for intestinal epithelium. Multidrug resistance-associated protein 4 (MRP4) plays an important role in renal secretion of adefovir. Immunofluorescence images showed that MRP4 is localized in the basolateral membrane of Caco-2 cells. This localization was further confirmed by Western blotting of the apical and basolateral membrane fractions that were isolated by a novel method involving biotinylation of respective membrane proteins and affinity enrichment. MRP4-knockdown Caco-2 cells were produced by stable transfection with MRP4-specific siRNA expression plasmid. These cells showed reduced MRP4 protein expression and corresponding reduction in the basolateral egress of adefovir when adefovir dipivoxil was dosed on the apical side. A comparison of these data with the reduction in the basolateral egress of adefovir by the general MRP inhibitor indomethacin established that MRP4, among MRPs, plays a predominant role in the basolateral egress of adefovir in Caco-2 cells. The results highlight the importance of MRP4 in oral absorption of adefovir dipivoxil, and suggest that significant drug-drug interactions can occur if an MRP4 inhibitor is co-administered with adefovir dipivoxil.     

Transport characteristics of peptides and peptidomimetics: II. Hydroxyethylamine bioisostere‐containing peptidomimetics as substrates for the oligopeptide transporter and P‐glycoprotein in the intestinal mucosa

Abstract: Peptide bond bioisosteres, such as hydroxyethylamine (Hea), have frequently been used to stabilize metabolically labile peptide bonds in peptidomimetic drug design in an effort to increase the oral bioavailability of drug candidates. However, the impact of the peptide bond bioisosteres on the cell permeation characteristics of peptidomimetics is not well understood, particularly with respect to the effects on the substrate activity for proteins that can restrict (e.g. P‐glycoprotein, P‐gp) or facilitate (e.g. the oligopeptide transporter, OPT) intestinal mucosal permeation of peptidomimetics. In this study, terminally free and terminally modified (N‐acetylated and C‐amidated) peptidomimetics of H‐Ala‐Phe‐OH and H‐Ala‐Phe‐Ala‐OH with the Ala‐Phe peptide bonds replaced by Hea bioisosteres were synthesized. Transport characteristics of these peptidomimetics were investigated using Caco‐2 cell monolayers as an in vitro model of the intestinal mucosa. The study showed that the Hea bioisostere stabilized the peptidomimetics to protease metabolism in Caco‐2 cells. All terminally free peptidomimetics showed significant affinity and substrate activity for OPT. The affinity and substrate activity for OPT were stereoselective for peptidomimetics containing an S,S‐configuration for the two adjacent chiral centers related to the Hea bioisostere. Three of the four terminally modified peptidomimetics showed significant substrate activity for P‐gp and, interestingly, the substrate activity for P‐gp was also stereoselective; however, it was in favor of an R,R‐configuration for the two adjacent chiral centers related to the Hea bioisostere.     

Evaluation of creatine transport using Caco-2 monolayers as an in vitro model for intestinal absorption.

Creatine is a nutraceutical that has gained popularity in both well-trained and casual athletes for its performance-enhancing or ergogenic properties. The major disadvantages of creatine monohydrate formulations are poor solubility and oral bioavailability. In the present study, creatine transport was examined using Caco-2 monolayers as an in vitro model for intestinal absorption. Confluent monolayers of Caco-2 cells (passage 25-35) were used for the permeability studies. Monolayers were placed in side-by-side diffusion chambers. (14)C-Creatine (0.1-0.5 microCi/mL) was added to either the apical or basolateral side, and the transport of the creatine across the Caco-2 monolayer was measured over a 90-min period. The apical to basolateral transport of (14)C-creatine was small, ranging from 0.2-3% of the original amount appearing on the receiver side in a 90-min period. Interestingly, the basolateral to apical permeability of radiolabeled creatine was substantially greater than that observed in the apical to basolateral direction. Studies with drug efflux transport inhibitors indicate that neither the P-glycoprotein nor multidrug resistance-associated protein is involved in the enhanced basolateral to apical transport of creatine.     

Analysis of the Complicated Nonlinear Pharmacokinetics of Orally Administered Telmisartan in Rats Using a Stable Isotope-IV Method

This study aimed to kinetically analyze the nonlinear absorption and systemic exposure of telmisartan (TEL) after oral administration to rats by using a stable isotope-IV method. Rats were orally administered different dose of TEL, followed by the intravenous injection of 0.005 mg/kg of deuterium-labeled TEL (TEL-d3). Assuming that TEL-d3 shows same pharmacokinetic properties with TEL, systemic clearance (CL_tot), oral bioavailability (F_oral), and intestinal and hepatic availability (F_a*F_g, F_h) of TEL were calculated in each individual rat. AUC_po of TEL increased disproportionately with dose and showed a sigmoid-type relation, indicating the involvement of multi-nonlinear processes in oral absorption of TEL. F_a*F_g of TEL increased with dose at the low-dose range while decreased at the high-dose range. In contrast, F_h increased and CL_tot decreased significantly in the middle range (2 to 6 mg/kg). As main factors of nonlinearity, saturations of solubility, efflux transport in the intestine, and the hepatic uptake of TEL were indicated. In conclusion, this study demonstrated a high possibility of a stable isotope-IV method to characterize complicated pharmacokinetic properties of oral drugs in animals, which can help to consider the future risks in their clinical use.