Active intestinal absorption of fluoroquinolone antibacterial agent ciprofloxacin by organic anion transporting polypeptide, Oatp1a5

Fluoroquinolone antimicrobial drugs are absorbed efficiently after oral administration despite of their hydrophilic nature, implying an involvement of carrier‐mediated transport in their membrane transport process. It has been that several fluoroquinolones are substrates of organic anion transporter polypeptides OATP1A2 expressed in human intestine derived Caco‐2 cells. In the present study, to clarify the involvement of OATP in intestinal absorption of ciprofloxacin, the contribution of Oatp1a5, which is expressed at the apical membranes of rat enterocytes, to intestinal absorption of ciprofloxacin was investigated in rats. The intestinal membrane permeability of ciprofloxacin was measured by in situ and the vascular perfused closed loop methods. The disappeared and absorbed amount of ciprofloxacin from the intestinal lumen were increased markedly in the presence of 7,8‐benzoflavone, a breast cancer resistance protein inhibitor, and ivermectin, a P‐glycoprotein inhibitor, while it was decreased significantly in the presence of these inhibitors in combination with naringin, an Oatp1a5 inhibitor. Furthermore, the Oatp1a5‐mediated uptake of ciprofloxacin was saturable with a K_m value of 140 µm , and naringin inhibited the uptake with an IC₅₀ value of 18 µm by Xenopus oocytes expressing Oatp1a5. Naringin reduced the permeation of ciprofloxacin from the mucosal‐to‐serosal side, with an IC₅₀ value of 7.5 µm by the Ussing‐type chamber method. The estimated IC₅₀ values were comparable to that of Oatp1a5. These data suggest that Oatp1a5 is partially responsible for the intestinal absorption of ciprofloxacin. In conclusion, the intestinal absorption of ciprofloxacin could be affected by influx transporters such as Oatp1a5 as well as the efflux transporters such as P‐gp and Bcrp. Copyright © 2012 John Wiley & Sons, Ltd.     

Saturable Absorptive Transport of the Hydrophilic Organic Cation Ranitidine in Caco-2 Cells: Role of pH-Dependent Organic Cation Uptake System and P-Glycoprotein

Purpose The purpose of this work was to investigate the involvement of carrier-mediated apical (AP) uptake and efflux mechanisms in the absorptive intestinal transport of the hydrophilic cationic drug ranitidine in Caco-2 cells. Methods Absorptive transport and AP uptake of ranitidine were determined in Caco-2 cells as a function of concentration. Permeability of ranitidine in the absorptive and secretory directions was assessed in the absence or presence of the P-glycoprotein (P-gp) inhibitor, GW918. Characterization of the uptake mechanism was performed with respect to inhibitor specificity, pH, energy, membrane potential, and Na⁺ dependence. Efflux from preloaded monolayers was evaluated over a range of concentrations and in the absence or presence of high extracellular ranitidine concentrations. Results Saturable absorptive transport and AP uptake of ranitidine were observed with K_m values of 0.27 and 0.45 mM, respectively. The ranitidine absorptive permeability increased and secretory permeability decreased upon inhibition of P-gp. AP ranitidine uptake was inhibited in a concentration-dependent fashion by a diverse set of organic cations including tetraethylammonium, 1-methyl-4-phenylpyridinium, famotidine, and quinidine. AP ranitidine uptake was pH and membrane potential dependent and reduced under conditions that deplete metabolic energy. Efflux of [³H]ranitidine across the basolateral membrane was neither saturable as a function of concentration nor trans stimulated by unlabeled ranitidine. Conclusions Saturable absorptive transport of ranitidine in Caco-2 cells is partially mediated via a pH-dependent uptake transporter for organic cations and is subject to attenuation by P-gp. Inhibition and driving force studies suggest the uptake carrier exhibits similar properties to cloned human organic cation transporters. The results also imply ranitidine transport is not solely restricted to the paracellular space.     

Characterization of Calu-3 cell monolayers as a model of bronchial epithelial transport: organic cation interaction studies

Background: To fully exploit organic cation transporters for targeted drug delivery in the lung, the use of a readily available and well-characterized tissue culture model and cheap easily detectable substrates is indispensable.

Objectives: To investigate the suitability of Calu-3 as tissue model for characterizing organic cation permeation across the bronchial cells using a fluorescent dye, 4-(4-(Dimethylamino)styryl)-N-methylpyridinium iodide (4-DI-1-ASP).

Methods: Substrate uptake, inhibition, and transport were performed to establish active transport mechanism. Organic cation transporter expression was determined with quantitative polymerase chain reaction (qPCR), immune-histochemistry, and fluorescent microscopy.

Results: 4-Di-1-ASP uptake in Calu-3 cells was concentration (K_m = 2.7?±?0.3?mM, V_max = 4.6?±?2.6 nmol/µg protein/30?min), temperature (uptake at 37°C>>4°C), and pH dependent (higher uptake at pH ≥ 7). L-carnitine, verapamil, and corticosterone significantly inhibited its uptake with IC₅₀ of 28.2, 0.81, and 0.12?mM, respectively. Transport of the dye across the cells was polarized (AP→BL transport was 2.5-fold > BL→AP), saturable (Km = 43.9?±?3.2) (µM; Vmax =0.0228± nmol/cm²/sec) and reduced 3-fold by metabolic inhibition. The expression pattern of the organic cation transporters (OCT) and carnitine/organic cation transporter (OCTN) isoforms was: OCT1<<OCT3 <OCTN1<OCTN2; OCT2 was not detected.

Conclusions: Based on qPCR, immunohistochemistry, uptake and transport data, the Calu-3 cells can be used as a model for not only studying strategies for optimizing the effect of inhaled organic cations, but also for cross-validating newly-developed respiratory cell lines.     

Membrane transport mechanisms of choline in human intestinal epithelial LS180 cells

The aim of the present study was to investigate the membrane transport mechanisms of choline using human intestinal epithelial LS180 cells. The mRNA of choline transporter‐like proteins (CTLs) was expressed significantly in LS180 cells, and the rank order was CTL1 > CTL4 > CTL3 > CTL2 > CTL5. In contrast, the mRNA expression of other choline transporters, organic cation transporter (OCT) 1, OCT2 and high‐affinity choline transporter 1 (CHT1), was considerably lower in LS180 cells. Five mm unlabelled choline, hemicolinium‐3 and guanidine, but not tetraethylammonium, inhibited the cellular uptake of 100 µm choline in LS180 cells. The uptake of choline into LS180 cells was virtually Na⁺‐independent. The uptake of choline was significantly decreased by acidification of the extracellular pH; however, it was not increased by alkalization of the extracellular pH. In addition, both acidification and alkalization of intracellular pH decreased the uptake of choline, indicating that the choline uptake in LS180 cells is not stimulated by the outward H⁺ gradient. On the other hand, the uptake of choline was decreased by membrane depolarization along with increasing extracellular K⁺ concentration. In addition, the Na⁺‐independent uptake of choline was saturable, and the K_m value was estimated to be 108 µm . These findings suggest that the uptake of choline into LS180 cells is membrane potential‐dependent, but not outward H⁺ gradient‐dependent. Copyright © 2014 John Wiley & Sons, Ltd.     

Disposition kinetics of a dipeptide ester prodrug of acyclovir and its metabolites following intravenous and oral administrations in rat

The objective of this work was to study the disposition kinetics of valine-valine–acyclovir (VVACV), a dipeptide ester prodrug of acyclovir following intravenous and oral administrations in rat. A validated LC-MS/MS analytical method was developed for the analysis VVACV, Valine-Acyclovir (VACV), and Acyclovir (ACV) using a linear Ion Trap Quadrupole. ACV was administered orally for comparison purpose. In the VVACV group, both blood and urine samples and in the ACV group only blood samples were collected. All the samples were analyzed using LC-MS/MS. The LLOQ for ACV, VACV, and VVACV were 10, 10, and 50?ng/ml, respectively. Relevant pharmacokinetic parameters were obtained by non-compartmental analyses of data with WinNonlin. Following i.v. administration of VVACV, AUC_0-inf (min*μM) values for VVACV, VACV, and ACV were 55.06, 106, and 466.96, respectively. The AUC obtained after oral administration of ACV was 178.8. However, following oral administration of VVACV, AUC_0-inf values for VACV and ACV were 89.28 and 810.77, respectively. Thus the exposure of ACV obtained following oral administration of VVACV was almost 6-fold higher than ACV. This preclinical pharmacokinetic data revealed that VVACV has certainly improved the oral bioavailability of ACV and is an effective prodrug for oral delivery of ACV.     

Mechanism of Corneal Permeation of L-Valyl Ester of Acyclovir: Targeting the Oligopeptide Transporter on the Rabbit Cornea

Purpose. To delineate mechanisms associated with the corneal transport of a L-valine prodrug of an antiviral agent, acyclovir. Method. The permeability and enzymatic hydrolysis of L-Val-ACV were evaluated using freshly excised rabbit cornea. Transport mechanism across rabbit cornea was investigated through a competitive inhibition study of L-Val-ACV with other substrates of human peptide transporter (hPepT1). Results. L-Valyl ester of Acyclovir (L-Val-ACV) was approximately threefold more permeable across the intact rabbit cornea than acyclovir (ACV). Dipeptides, -lactam antibiotics, and angiotensin converting enzyme (ACE) inhibitors, strongly inhibited the transport of L-Val-ACV indicating that a carrier mediated transport system specific for peptides is primarily responsible for the corneal permeation of L-Val-ACV. L-Val-ACV transport was found to be saturable (K _m = 2.26 ± 0.34 mM, J _max = 1.087 ± 0.05 nmoles cm^{– 2} min^{– 1}), energy and pH dependent. Conclusions. Functional evidence of an oligopeptide transport system present on the rabbit cornea has been established. The peptide transporter on the corneal epithelium may be targeted to improve the ocular bioavailability of poorly absorbed drugs.     

Substrate Specificity and Mechanism of the Intestinal Clonidine Uptake by Caco-2 Cells

Purpose This study was performed to characterize the substrate specificity and mechanism of the intestinal clonidine transport. Methods Uptake of [³H]clonidine into Caco-2 cells was investigated. Interaction with drugs was studied in competition assays. Results Uptake of [³H]clonidine was linear for up to 2 min, Na⁺-independent, and insensitive to changes in membrane potential, but strongly H⁺-dependent. The uptake rate of clonidine was saturable with kinetic parameters of 0.5 ± 0.1 mM (K_t) and 16.6 ± 1.8 nmol/2 min per mg of protein (V_max) at an outside pH of 7.5. Many drugs such as clonidine, guanabenz, methamphetamine, imipramine, clomipramine, nortriptyline, quinine, xylazine, ephedrine, and diphenhydramine strongly inhibited the [³H]clonidine uptake with K_i values between 0.15 and 1 mM. Conclusions Clonidine is transported by a carrier-mediated process. Substrate specificity and mechanism are very similar to the transport described in blood–brain barrier endothelial cells. The transport characteristics do not correspond to carriers for organic cations of the SLC22 family or the choline transporters CHT1 and CLT1. The system might be identical to the H⁺/tertiary amine antiporter. It interacts with a large number of both hydrophilic and lipophilic cationic drugs, and also, interestingly, with opiates.     

Mechanism of Acyclovir Uptake in Rat Jejunum

The intestinal uptake mechanism of the purine analogue, acyclovir, was investigated in rat jejunum using in vitro and in situ methods. The pyrimidine, uracil, was used as a reference compound for carrier-mediated transport, while the purine analogue, caffeine, served as the reference compound for passive diffusion. With the in vitro intestinal ring method, acyclovir uptake was linear in the concentration range 0.01–5 mM. No significant competition for uptake was observed with uracil, 6-mercaptopurine, hypoxanthine, caffeine, or adenine. In addition, use of 2,4-dinitrophenol (DNP), ouabain, or K⁺ substituted buffer did not reduce the rate of acyclovir uptake. The in situ single-pass perfusion method yielded a wall permeability of 0.2, which did not vary consistently with increasing concentration. Coperfusion of acyclovir with DNP did not decrease the wall permeability. None of the data provided evidence of a carrier-mediated transport system, and it was concluded that the uptake mechanism of acyclovir in the rat jejunum is predominantly via passive diffusion.     

Characterization of loxoprofen transport in Caco‐2 cells: the involvement of a proton‐dependent transport system in the intestinal transport of loxoprofen

Loxoprofen, a propionate non‐steroidal anti‐inflammatory drug (NSAID), is used widely in East Asian countries. However, little is known about the transport mechanisms contributing to its intestinal absorption. The objectives of this study were to characterize the intestinal transport of loxoprofen using the human intestinal Caco‐2 cell model. The transport of loxoprofen was investigated in cellular uptake studies. The uptake of loxoprofen into Caco‐2 cells was pH‐ and concentration‐dependent, and was described by a Michaelis–Menten equation with passive diffusion (K_m: 4.8 mm , V_max: 142 nmol/mg protein/30 s, and K_d: 2.2 μl/mg protein/30 s). Moreover, the uptake of loxoprofen was inhibited by a typical monocarboxylate transporter (MCT) inhibitor as well as by various monocarboxylates. The uptake of [¹⁴C] l ‐lactic acid, a typical MCT substrate, in Caco‐2 cells was saturable with relatively high affinity for MCT. Because loxoprofen inhibited the uptake of [¹⁴C] l ‐lactic acid in a noncompetitive manner, it was unlikely that loxoprofen uptake was mediated by high‐affinity MCT(s). Our results suggest that transport of loxoprofen in Caco‐2 cells is, at least in part, mediated by a proton‐dependent transport system. Copyright © 2016 John Wiley & Sons, Ltd.     

5′-Amino Acid Esters of Antiviral Nucleosides, Acyclovir, and AZT Are Absorbed by the Intestinal PEPT1 Peptide Transporter

Purpose. General use of nucleoside analogues in the treatment of viral infections and cancer is often limited by poor oral absorption. Valacyclovir, a water soluble amino acid ester prodrug of acyclovir has been reported to increase the oral bioavailability of acyclovir but its absorption mechanism is unknown. This study characterized the intestinal absorption mechanism of 5-amino acid ester prodrugs of the antiviral drugs and examined the potential of amino acid esters as an effective strategy for improving oral drug absorption. Methods. Acyclovir (ACV) and Zidovudine (AZT) were selected as the different sugar-modified nucleo-side antiviral agents and synthesized to L-valyl esters of ACV and AZT (L-Val-ACV and L-Val-AZT), D-valyl ester of ACV (D-Val-ACV) and glycyl ester of ACV (Gly-ACV). The intestinal absorption mechanism of these 5-amino acid ester prodrugs was characterized in three different experimental systems; in siturat perfusion model, CHO/hPEPTl cells and Caco-2 cells. Results. Testing 5-amino acid ester prodrugs of acyclovir and AZT, we found that the prodrugs increased the intestinal permeability of the parent nucleoside analogue 3- to 10-fold. The dose- dependent permeation enhancement was selective for the L-amino acid esters. Competitive inhibition studies in rats and in CHO cells transfected with the human peptide transporter, hPEPTl, demonstrated that membrane transport of the prodrugs was mediated predominantly by the PEPT1 H⁺/dipeptide cotransporter even though these prodrugs did not possess a peptide bond. Finally, transport studies in Caco-2 cells confirmed that the 5-amino acid ester prodrugs enhanced the transcellular transport of the parent drug. Conclusions. This study demonstrates that L-amino acid-nucleoside chimeras can serve as prodrugs to enhance intestinal absorption via the PEPT1 transporter, providing a novel strategy for improving oral therapy of nucleoside drugs.     

Intestinal transporters for endogenic and pharmaceutical organic anions: the challenges of deriving in‐vitro kinetic parameters for the prediction of clinically relevant drug–drug interactions

Objectives This review provides an overview of intestinal human transporters for organic anions and stresses the need for standardization of the various in‐vitro methods presently employed in drug–drug interaction (DDI) investigations. Key findings Current knowledge on the intestinal expression of the apical sodium‐dependent bile acid transporter (ASBT), the breast cancer resistance protein (BCRP), the monocarboxylate transporters (MCT) 1, MCT3‐5, the multidrug resistance associated proteins (MRP) 1–6, the organic anion transporting polypetides (OATP) 2B1, 1A2, 3A1 and 4A1, and the organic solute transporter α/β (OSTα/β) has been covered along with an overview of their substrates and inhibitors. Furthermore, the many challenges in predicting clinically relevant DDIs from in‐vitro studies have been discussed with focus on intestinal transporters and the various methods for deducting in‐vitro parameters for transporters (K_m/K_i/IC50, efflux ratio). The applicability of using a cut‐off value (estimated based on the intestinal drug concentration divided by the K_i or IC50) has also been considered. Summary A re‐evaluation of the current approaches for the prediction of DDIs is necessary when considering the involvement of other transporters than P‐glycoprotein. Moreover, the interplay between various processes that a drug is subject to in‐vivo such as translocation by several transporters and dissolution should be considered.     

Involvement of an influx transporter in the blood–brain barrier transport of naloxone

Naloxone, a potent and specific opioid antagonist, has been shown in previous studies to have an influx clearance across the rat blood–brain barrier (BBB) two times greater than the efflux clearance. The purpose of the present study was to characterize the influx transport of naloxone across the rat BBB using the brain uptake index (BUI) method. The initial uptake rate of [³H]naloxone exhibited saturability in a concentration‐dependent manner (concentration range 0.5 µM to 15 mM ) in the presence of unlabeled naloxone. These results indicate that both passive diffusion and a carrier‐mediated transport mechanism are operating. The in vivo kinetic parameters were estimated as follows: the Michaelis constant, K_t, was 2.99±0.71 mM ; the maximum uptake rate, J_max, was 0.477±0.083 µmol/min/g brain; and the nonsaturable first‐order rate constant, K_d, was 0.160±0.044 ml/min/g brain. The uptake of [³H]naloxone by the rat brain increased as the pH of the injected solution was increased from 5.5 to 8.5 and was strongly inhibited by cationic H₁‐antagonists such as pyrilamine and diphenhydramine and cationic drugs such as lidocaine and propranolol. In contrast, the BBB transport of [³H]naloxone was not affected by any typical substrates for organic cation transport systems such as tetraethylammonium, ergothioneine or L ‐carnitine or substrates for organic anion transport systems such as p‐aminohippuric acid, benzylpenicillin or pravastatin. The present results suggest that a pH‐dependent and saturable influx transport system that is a selective transporter for cationic H₁‐antagonists is involved in the BBB transport of naloxone in the rat. Copyright © 2010 John Wiley & Sons, Ltd.     

Functional involvement of organic cation transporter1 (OCT1/Oct1) in the hepatic uptake of organic cations in humans and rats

The contribution of organic cation transporters to the saturable component in the hepatic uptake of 1-methyl-4-phenylpyridinium (MPP), tetraethylammonium (TEA), cimetidine, and metformin was examined by the use of human/rat organic cation transporter (hOCT1/rOct1)-expressing cells and human/rat hepatocytes. Transfection of rOct1 resulted in a considerable increase in the uptake of metformin, whereas that of hOCT1 resulted in only a slight increase. All test compounds (MPP, TEA, cimetidine, and metformin) accumulated in human and rat hepatocytes in a carrier-mediated manner. The K_m values for the uptake of MPP, TEA, cimetidine, and metformin into human and rat hepatocytes were comparable with those into hOCT1 and rOct1-expressing cells, respectively. In addition, the relative uptake activities, which were obtained by normalizing the intrinsic uptake clearances of TEA, cimetidine, and metformin against those values of MPP in human and rat hepatocytes, were similar with the uptake activities in hOCT1 and rOct1, respectively. These results suggest that the saturable component in the hepatic uptake of these cationic compounds may be mediated mainly by hOCT1/rOct1; therefore, it is meaningful to evaluate the saturable uptake profile of cationic compounds by the liver using both hOCT1/rOct1-expressing cells and human/rat hepatocytes.     

Contribution of organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 to hepatic uptake of nateglinide, and the prediction of drug-drug interactions via these transporters

Objectives We have investigated the contributions of organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 to the hepatic uptake of nateglinide, and the possibility of drug–drug interactions via these transporters. Methods Uptake studies using transporter‐expressing HEK293 cells and cryopreserved human hepatocytes were performed to examine the contributions of each transporter. Inhibition studies using cryopreserved human hepatocytes were performed to examine the possibility of drug–drug interactions. Key findings The rate of saturable hepatic uptake of nateglinide using human hepatocytes was 47.6%. A certain increase in uptake was observed in the examination using transporter‐expressing HEK293 cells, indicating contributions of OATP1B1 and OATP1B3 to hepatic nateglinide uptake. The 50% inhibitory concentration (IC50) values of nateglinide using cryopreserved human hepatocytes for uptake of estrone 3‐sulfate (substrate of OATP1B1), and cholecystokinin octapeptide (substrate of OATP1B3) were 168 and 17.4 µmol/l, respectively. Moreover, ciclosporin inhibited saturable hepatic uptake of nateglinide with an IC50 value of 6.05 µmol/l. The calculated 1 + I_in,max,u/IC50 values for inhibition of OATP1B1 and OATP1B3 by nateglinide, and the inhibition of saturable uptake of nateglinide by ciclosporin, were all close to 1, indicating a low clinical risk of drug–drug interaction with nateglinide taken up via OATP1B1 and OATP1B3. Conclusions OATP1B1 and OATP1B3 may have contributed to the hepatic uptake of nateglinide, but the possibility of drug–drug interactions appeared to be low.     

Proton-Cotransport of Pravastatin Across Intestinal Brush-Border Membrane

Purpose. The purpose of the present study is to clarify the intestinal brush-border transport mechanism of a weak organic acid, pravastatin, an HMG-CoA reductase inhibitor. Methods. The transport of pravastatin was studied by using intestinal brush-border membrane vesicles prepared from rabbit jejunum, and uptake by the membrane vesicles was measured using rapid filtration technique. Results. The initial uptake of [¹⁴C]pravastatin was markedly increased with decreases in extravesicular pH and showed a clear overshoot phenomenon in the presence of a proton gradient (pH_in/out = 7.5/5.5). A protonophore, carbonylcyanide p-trifluoromethoxyphenylhydrazone, significantly reduced the uptake of [¹⁴C]pravastatin. In addition, an ionophore for sodium, potassium and proton, nigericin, stimulated the uptake of [¹⁴C]pravastatin in the presence of a potassium gradient ([K ⁺ ]_in/[K⁺ ]_out = 0/145 mM). On the other hand, neither the imposition of an inwardly directed sodium gradient nor an outwardly directed bicarbonate gradient stimulated the uptake of [¹⁴C]pravastatin. In the presence of a proton gradient (pH_in/out = 7.5/5.5), the initial uptake of pravastatin was saturable with the apparent K_t of 15.2 ± 3.2 mM and J_max of 10.6 ± 1.21 nmol/mg protein/10 sec. The uptake of pravastatin was significantly inhibited by monocarboxylic acid compounds such as acetic acid and nicotinic acid in a competitive manner but not by di- or tri-carboxylic acids, or acidic amino acid. Conclusions. It was concluded that a pH-dependent transport of pravastatin across the brush-border membrane occurs by a proton-gradient dependent carrier-mediated mechanism rather than by simple diffusion of its unionized form.     

Acyclovir Permeation Enhancement Across Intestinal and Nasal Mucosae by Bile Salt-Acylcarnitine Mixed Micelles

The purpose of this study was to investigate the absorption enhancement of acyclovir, an antiviral agent, by means of bile salt-acylcarnitine mixed micelles. The specificity, site dependence, palmitoyl-DL-carnitine chloride (PCC) concentration dependence, and effects of absorption promoters on acyclovir absorption via the nasal cavity (N) and four different intestinal segments of the rat, i.e., duodenum (D), upper jejunum (UJ), combined lower jejunum and ileum (LJ), and colon (C) were evaluated. The present study employed the rat in situ nasal and intestinal perfusion techniques and utilized sodium glycocholate (NaGC), three acylcarnitines, and their mixed micelles as potential nasal and intestinal absorption promoters. Acylcarnitines used were DL-octanoylcarnitine chloride (OCC), palmitoyl-DL-carnitine chloride (PCC), and DL-stearoylcarnitine chloride (SCC). All acylcarnitines and NaGC by themselves produced negligible enhancement of acyclovir absorption in the rat intestine, while OCC and SCC were totally ineffective in the nasal cavity. However, the mixed micellar solutions of NaGC with PCC or SCC could significantly increase the mucosal membrane permeability of acyclovir in the colon and nasal cavity. On the other hand, NaGC-OCC mixed micelles slightly increased the absorption of acyclovir by both routes. When a mixed micellar solution of NaGC with PCC was used, the rank order of apparent acyclovir permeability (P_app; cm/sec), corrected for surface area of absorption, was N (10.54 ± 0.62 x 10^–5) > D (6.82 ± 0.30 x 10^–5) > LJ (2.90 ± 0.08 x 10^–5) > C (2.54 ± 0.14 x 10^–5) > UJ (2.30 ± 0.22 x 10^–5). In contrast, the P_app rank order for acyclovir without any absorption promoter was D (2.49 ± 0.44 x 10^–5) > UJ (0.64 ± 0.03 x 10^–5) > LJ, C, and N (0). The effect of mixed micellar solutions was synergistic and was much greater than that with single adjuvants probably because of micellar solubilization of acylcarnitines by NaGC. The magnitude of absorption promotion was dependent on the hydrophobicity, i.e., carbon-chain length of the acylcarnitines. The enhanced permeability could be reversed within 60-120 min after removal of the adjuvant from the duodenum, colon, and nasal cavity. These results suggest that bile salt-acylcarnitine mixed micelles can be used as intestinal or nasal mucosal absorption promoters of poorly permeable agents.     

Plant polyphenols and multidrug resistance: Effects of dietary flavonoids on drug transporters in Caco-2 and MDCKII-MDR1 cell transport models

The hypothesis tested was that specific flavonoids such as epicatechin gallate, epigallocatechin gallate, genistein, genistin, naringenin, naringin, quercetin and xanthohumol will modulate cellular uptake and permeability (P_e) of multidrug-resistant substrates, cyclosporin A (CSA) and digoxin, across Caco-2 and MDCKII-MDR1 cell transport models. ³H-CSA/³H-digoxin transport and uptake experiments were performed with and without co-exposure of the flavonoids. Aglycone flavonoids reduced the P_e of CSA to a greater extent than glycosylated flavonoids with 30?µM xanthohumol producing the greatest effect (7.2?×?10^–6 to 6.6?×?10^–7 and 17.9?×?10^–6 to 4.02?×?10^–6?cm?s^–1 in Caco-2 and MDCKII-MDR1 cells, respectively); while no measurable effects were seen with digoxin. Xanthohumol significantly demonstrated (1) saturable efflux, (2) increased uptake of ³H-digoxin and (3) decreased uptake of ³H-CSA in the Caco-2 cells. The transport data suggests that xanthohumol effects transport of CSA in a manner that is distinct from the digoxin efflux pathway and suggests that intestinal transport of these MDR1 substrates is more complex than previously reported.     

Transbuccal Delivery of Acyclovir: I. In Vitro Determination of Routes of Buccal Transport

Purpose. To determine the major routes of buccal transport of acyclovir and to examine the effects of pH and permeation enhancer on drug permeation. Methods. Permeation of acyclovir across porcine buccal mucosa was studied by using side-by-side flow through diffusion cells at 37°C. The permeability of acyclovir was determined at pH range of 3.3 to 8.8. Permeability of different ionic species was calculated by fitting the permeation data to a mathematical model. Acyclovir was quantified using HPLC. Results. Higher steady state fluxes were observed at pH 3.3 and 8.8. The partition coefficient (1-octanol/buffer) and the solubility of acyclovir showed the same pH dependent profile as that of drug permeation. In the presence of sodium glycocholate (NaGC) (2–100 mM), the permeability of acyclovir across buccal mucosa was increased 2 to 9 times. This enhancement was independent of pH and reached a plateau above the critical micelle concentration of NaGC. The permeabilities of anionic, cationic, and zwitterionic species were 3.83 × 10^–5, 4.33 × 10^–5, and 6.24 × 10^–6cm/sec, respectively. Conclusions. The in vitropermeability of acyclovir across porcine buccal mucosa and the octanol-water partitioning of the drug were pH dependent. A model of the paracellular permeation of the anionic, cationic, and zwitterionic forms of acyclovir is consistent with these data. The paracellular route was the primary route of buccal transport of acyclovir, and the enhancement of transbuccal transport of acyclovir by sodium glycocholate (NaGC) appeared to operate via this paracellular route.     

Suitability of Enalapril as a Probe of the Dipeptide Transporter System: In Vitro and In Vivo Studies

Purpose. Previous in situ and in vitro studies indicated that the intestinal absorption of enalapril is a saturable carrier-mediated process via the dipeptide transporter system (DTS); however, the oral absorption of enalapril has not been reported to be a saturable process in vivo. Our objectives were to: 1) evaluate the suitability of enalapril as a probe of the DTS, and 2) compare various experimental models as they pertain to studying the DTS. Methods. The in vitro uptake of enalapril by rat intestinal rings and permeability across Caco-2 cells were studied as a function of concentration and in the presence of compounds that are known substrates of the DTS. The effect of enalapril on the uptake of [³H]-glycyl-L-proline (gly-L-pro) by Caco-2 cells was also examined. In vivo studies were conducted in rats (1 to 50 mg/kg) and dogs (0.06 to 6 mg/kg) to evaluate the oral absorption of enalapril over a wide dose range. Results. In vitro intestinal uptake/permeability of enalapril was not saturable nor inhibited by p-lactam antibiotics, gly-L-pro, or SQ-29852. Moreover, a 20,000-fold molar excess of enalapril did not inhibit the uptake of [³H]-gly-L-pro by Caco-2 cells. The in vivo studies in rats and dogs did not demonstrate saturable absorption. Conclusions. The present in vitro and in vivo results indicated that enalapril is primarily absorbed by a non-saturable, passive diffusion process and it is not a suitable model compound for studying the DTS.     

The Transport Mechanism of an Organic Cation,Disopyramide, by Brush-border Membranes. Comparison Between Renal Cortex and Small Intestine of the Rat

Abstract— The characteristics of disopyramide uptake in brush-border membrane vesicles isolated from rat renal cortex and small intestine were investigated. Transport of disopyramide into an osmotically reactive intravesicular space was observed with notable binding to the membrane surface. An outwardly directed H⁺ gradient stimulated disopyramide uptake, resulting in a transient uphill transport in both brush-border membranes. As for the renal brush-border membrane, the H⁺ gradient itself appeared to be the driving force for this stimulation of uptake. These findings suggest that disopyramide-H⁺ antiport is the mechanism of disopyramide action in renal cell membrane. The initial uptake was saturable (K_m and V_max of 680 μm and 1·25 nmol (mg protein)^?1/30 s, respectively). The stimulation of disopyramide uptake by an outward H⁺ gradient in rat intestinal brush-border membrane was due to an interior negative H⁺-diffusion potential. A K⁺-diffusion potential (interior negative) enhanced disopyramide uptake. These results suggest that there are different mechanisms of disopyramide uptake for renal and intestinal brush-border membrane vesicles.