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Objective: Ifosfamide (IFO) is considered an essential drug for the treatment of pediatric, adolescent and young adult patients with solid tumors. Hemorrhagic cystitis (HC) is one of the dose-limiting toxicity of IFO. However, there are insufficient evidence for risk factor and supportive care of IFO-induced HC., Methods: In this retrospective study, patients (<30-year-old) with malignant solid tumors who had been treated with IFO-based chemotherapy, were categorized according to the presence or absence of HC, and were analyzed possible risk factors for IFO-induced HC. In our institution, continuous hydration to increase urine output and intravenous 2-mercaptethane sulfonate (mesna) are used for prophylaxis of IFO-induced HC. Increased hydration and dosage of mesna are administered to patients who develop IFO-induced HC; they also receive 24-h continuous infusion of mesna in subsequent treatment cycles., Results: Nine treatment regimens were used in the 70 study patients. The range of daily IFO dosage was 1.2-3.0 g/m(2). HC occurred in 14/425 IFO-based chemotherapy cycles (3.3%). The daily IFO dosages (mean ± SD) in patients with or without HC were 2.23 ± 0.58 g/m(2) and 1.85 ± 0.50 g/m(2), respectively (P = 0.006). Only one of the nine patients who developed IFO-induced HC had experienced this complication in a subsequent cycle of treatment., Conclusion: The incidence of IFO-induced HC may be associated with the dosage of IFO. When administering IFO higher than 2.0 g/m(2)/day, the volume of hydration, dosage of mesna and duration of mesna infusion should be increased to prevent HC., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: It has been reported that intestinal urate excretion is increased at chronic kidney disease (CKD) state. In this report, whether uremic toxins are involved in the upregulation of intestinal breast cancer resistance protein (BCRP), an intestinal urate exporter, was examined., Methods: Uremic toxins that were increased at least 15-fold at CKD state were selected for investigation. Caco-2 cells were exposed to these uremic toxins at clinically relevant concentrations. mRNA was quantified by real-time PCR, and flow cytometry was utilized to measure BCRP protein and function in Caco-2 cells. Transcellular secretory transport of [(14) C]urate was determined utilizing Transwell studies after uremic toxin exposure., Key Findings: Indoxyl sulfate (IS) treatment alone resulted in ∼ 3-fold increase in BCRP mRNA in Caco-2 cells. Membrane protein expression of BCRP in Caco-2 cells also was increased by 1.8-fold after treatment with IS. Intracellular accumulation of pheophorbide A, a selective BCRP substrate, was decreased by 22% after IS treatment for 3 days. Consistent with these findings, transcellular secretory transport of urate across Caco-2 cell monolayers was increased by 22%., Conclusion: Intestinal urate secretion may be increased at CKD state partially by upregulation of intestinal BCRP by uremic toxins such as IS., (© 2014 Royal Pharmaceutical Society.)

Background: Serum urate (SUA) level is affected by alteration in urinary reabsorption caused by clinically important drugs; however, there are no experimental models suitable to assess their effect on renal reabsorption. We, therefore, aimed to establish an experimental system co-expressing the urate transporters URAT1 (SLC22A12) and URATv1 (SLC2A9) (designated UUv cells) at the apical and basolateral membranes, respectively., Methods: Apical uptake and vectorial transport of [(14)C]urate in the apical-to-basolateral direction in UUv cells were measured in the presence or absence of uricosuric benzbromarone or anti-uricosuric trans-stimulators., Results: The urate permeability in the apical-to-basolateral direction remarkably increased by 7.0-fold in UUv cells, compared with non-transfected mock cells. The apical-to-basolateral transport was cis-inhibited by benzbromarone, but trans-stimulated by pyrazinecarboxylic acid and monocarboxylates such as nicotinate and lactate. Furthermore, salicylate showed both trans-stimulation and cis-inhibition in the urate transport at low and high concentrations, respectively. Finally, coexpression of URAT1 and URATv1 in human kidney epithelial cells was exhibited immunohistochemically., Conclusions: It is demonstrated that functional cooperation of URAT1 and URATv1 is essential for renal reabsorption of urate, and in the established system influence of drugs on SUA is reflected in the alteration of urate permeability across the renal tubular epithelial cells.

The aim of this study was to develop and validate an assay based on liquid chromatography-tandem mass spectrometry to quantitate talinolol in rat plasma. After a simple protein precipitation step, separation was performed by reversed-phase liquid chromatography using gradient elution with acetonitrile-water-formic acid. Electrospray ionization in the positive ion mode with multiple reaction monitoring was used to analyze talinolol employing propranolol as internal standard. The calibration curve for talinolol was linear over the concentration range 1-250 ng/mL with a correlation coefficient > 0.995. The method was sensitive (limit of quantitation, 1 ng/mL) and had acceptable accuracy (85-115% of true values) and precision (intra- and inter-assay CV < 15%). Recovery of talinolol at concentrations of 1, 25, and 250 ng/mL was >78%. The method was successfully applied to the determination of the oral pharmacokinetics of talinolol in rat.

The objective of this study was to examine the transport activity of the human organic anion transporter OATP-C (SLC21A6) for oligopeptides that are eliminated rapidly from the systemic circulation. We focused on an opioid peptide analogue, [D-Ala(2), D-Leu(5)]-enkephalin (DADLE), a linear pentapeptide modified to be stable. [(3)H]DADLE was taken up by rat isolated hepatocytes in a saturable manner and highly accumulated in the liver after intravenous administration to rats. The uptake of [(3)H]DADLE by the isolated hepatocytes was inhibited by several organic anions and pentapeptides, but not by tetra- or tripeptides. When OATP-C was expressed in Xenopus laevis oocytes, a significant increase in uptake of [(3)H]DADLE was observed. Moreover, the inhibitory effects of various compounds, including some peptides, on [(3)H]estrone-3-sulfate uptake by OATP-C were similar to those observed in [(3)H]DADLE uptake by rat isolated hepatocytes. In conclusion, it was demonstrated that OATP-C contributes to the rapid hepatic excretion of peptides and peptide-mimetic drugs.

Transport of quinolone antimicrobials and the contribution of the secretory transporter P-glycoprotein were studied in-vivo and in-vitro. In rat intestinal tissue (Ussing chambers method) and human Caco-2 cells (Transwell method), grepafloxacin showed secretory-directed transport. In both experimental systems, the secretory-directed transport was decreased by ciclosporin A, an inhibitor of P-glycoprotein, and probenecid, an inhibitor of anion transport systems. This suggested the contribution of P-glycoprotein and anion-sensitive transporter(s). The involvement of P-glycoprotein was investigated by using a P-glycoprotein over-expressing cell line, LLC-GA5-COL150, and P-glycoprotein-gene-deficient mice (mdr1a(-/-)/1b(-/-) mice). LLC-GA5-COL150 cells showed secretory-directed transport of grepafloxacin, while the parent cell line, LLC-PK1, did not. The secretory-directed transport of sparfloxacin and levofloxacin was also detected in LLC-GA5-COL150 cells. In the mdr1a(-/-)/1b(-/-) mice, the intestinal secretory clearance was smaller than that in wild-type mice after intravenous administration of grepafloxacin. Moreover, the absorption from an intestinal loop in mdr1a(-/-)/1b(-/-) mice was larger than that in wild-type mice. Accordingly, it appears that some quinolones are transported by secretory transporters, including P-glycoprotein. The involved transporters function in-vivo not only to transport grepafloxacin from blood to intestine but also to limit its intestinal absorption.

We present here the evidence of molecular and functional expression of LAT1 and LAT2, subunits of the large neutral amino acid transporter system L, in cultured brain capillary endothelial cells of the rat. By means of the RT-PCR method, transcripts of LAT1, LAT2 and heavy chain of 4F2 antigen (4F2hc) were detected in rat primary cultured brain capillary endothelial cells (BCECs) and immortalized subline, RBEC1. The uptake properties of RBEC1, such as [3H]leucine and L-[3H]DOPA uptake, were similar to those of primary cultured BCECs. So, RBEC1 may retain almost native properties of the large neutral amino acid uptake activities. [3H]Leucine uptake by RBEC1 showed two saturable components and the Km values of the high- and low-affinity components were 8.92+/-3.18 and 119+/-45 microM, respectively. The Km value of the high-affinity component agreed well with that of LAT1 and the amino acid transport selectivity of RBEC1 was similar to that of LAT1. Therefore, it is suggested that LAT1 is important at the blood-brain barrier of rats. Additionally, the Km value of the low-affinity component was similar to that of LAT2. These observations indicate that LAT1 and LAT2 are involved as transporters for large neutral amino acids at the blood-brain barrier. Additionally, we concluded that RBEC1 is useful as an in-vitro model for evaluation of the pharmacological relevance of system L at the blood-brain barrier.

We pharmacokinetically examined the effect of gamma-butyrobetaine, a precursor of L-carnitine, on the change of fatty acid metabolism in juvenile visceral steatosis (JVS) mice, which have systemic L-carnitine deficiency due to lack of L-carnitine transporter activity. The concentrations of total free fatty acid (FFA), palmitic acid and stearic acid in the liver of JVS mice were significantly higher than those in wild-type mice. After intravenous administration of gamma-butyrobetaine (50 mg kg(-1)), the concentration of L-carnitine in the plasma of JVS mice reached about twice that of the control level and levels in the brain, liver and kidney were also significantly increased, whereas those in wild-type mice hardly changed. Although the plasma concentrations of FFA in both types of mice were unchanged after administration of gamma-butyrobetaine, the concentrations of palmitic acid and stearic acid were significantly decreased. In particular, the liver concentration of FFA in JVS mice was decreased to the wild-type control level, accompanied by significant decreases in long-chain fatty acids, palmitic acid and stearic acid, whereas those in wild-type mice were not changed. These results suggest that gamma-butyrobetaine can be taken up into organs, including the liver, of JVS mice, and transformed to L-carnitine. Consequently, administration of gamma-butyrobetaine may be more useful than that of L-carnitine itself for treatment of primary deficiency of carnitine due to a functional defect of the carnitine transporter.

The intestinal transport of an organic anion, p-aminohippuric acid (PAH), was studied in Caco-2 cell monolayers and rat intestinal tissue mounted in Ussing chambers. In both experimental methods, PAH exhibited vectorial transport with significantly greater permeability in the secretory direction than the absorptive direction, indicating net secretion. This secretory transport required metabolic energy, but protons or hydroxyl ions were not involved as the driving force. In Caco-2 monolayers, secretory transport of [3H]PAH was decreased, and the intracellular accumulation of PAH was increased with increasing concentration of unlabelled PAH at the basolateral side. Addition of probenecid and genistein at the basolateral side decreased the secretory transport of [3H]PAH; the accumulation was not changed by probenecid, but was increased by genistein. In addition, the initial uptake rate of [3H]PAH from the basolateral side was decreased by both PAH and probenecid, but not by genistein. Therefore, it is suggested that the transport of PAH in Caco-2 cells is regulated by several transporters: a genistein-sensitive transporter on the apical membrane and probenecid-sensitive transporters on both the basolateral and apical membranes. In rat intestinal tissues, the transport rate of PAH showed regional variation (ileum > jejunum > duodenum), suggesting that secretory transporters with high activity exist predominantly in the lower region of the small intestine. The results suggest that PAH transport in both Caco-2 cells and rat intestinal tissues is regulated by multiple transporters on the apical and basolateral membranes, and these transporters have different characteristics.

The participation of the monocarboxylic acid transporter MCT1 in the intestinal absorption of weak organic acids has been clarified by functional characterization, by use of stably transfected cells, and by immunohistochemical location of the transporter in intestinal tissues. Immunohistochemical analysis by use of the anti-MCT1 antibody showed that MCT1 is distributed throughout the upper and lower intestines, especially in the basolateral membrane and, to a lesser extent, in the brush-border membrane. When the transporter gene rat MCT1 was transfected into MDA-MB231 cells, transport of benzoic acid, a model weak organic acid that has been generally believed to be transported across the cell membranes by passive diffusion, and lactic acid in rat MCT1-transfected cells was significantly increased compared with transport in cells transfected with the expression vector pRc-CMV alone (mock cells). The observed transport was pH-dependent and activity increased between pH 7.5 and pH 5.5, whereas pH-dependence in mock cells was moderate. Rat MCT1-mediated benzoic acid uptake was saturable, with an apparent Km value of 3.05 mM. In addition, MCT1 increased the efflux of [14C]benzoic acid from the cells. Several weak organic acids were also transported by rat MCT1. These results show that pH-dependent intestinal absorption of weak organic acids, previously explained in terms of passive diffusion according to the pH-partition hypothesis, is at least partially accounted for by MCT1-mediated transport energized at acidic pH by utilization of the proton gradient as a driving force.

The recently cloned organic cation transporter, OCTN2, isolated as a homologue of OCTN1, has been shown to be of physiological importance in the renal tubular reabsorption of filtered L-carnitine as a high-affinity Na+ carnitine transporter in man. Although the mutation of the OCTN2 gene has been proved to be directly related to primary carnitine deficiency, there is little information about the L-carnitine transport system in the liver. In this study, the characteristics of L-carnitine transport into hepatocytes were studied by use of cultured human hepatoma HLF cells, which expressed OCTN2 mRNA to a greater extent than OCTN1 mRNA. The uptake of L-carnitine into HLF cells was saturable and the Eadie-Hofstee plot showed two distinct components. The apparent Michaelis constant and the maximum transport rate were 6.59+/-1.85 microM (mean+/-s.d.) and 78.5+/-21.4 pmol/5 min/10(6) cells, respectively, for high-affinity uptake, and 590+/-134 microM and 1507+/-142 pmol/5 min/10(6) cells, respectively, for low-affinity uptake. The high affinity L-carnitine transporter was significantly inhibited by metabolic inhibitors (sodium azide, dinitrophenol, iodoacetic acid) and at low temperature (4 degrees C). Uptake of [3H]L-carnitine also required the presence of Na+ ions in the external medium. The uptake activity was highest at pH 7.4, and was significantly lower at acidic or basic pH. L-Carnitine analogues (D-carnitine, L-acetylcarnitine and gamma-butyrobetaine) strongly inhibited uptake of [3H] L-carnitine, whereas beta-alanine, glycine, choline, acetylcholine and an organic anion and cation had little or no inhibitory effect. In conclusion, L-carnitine is absorbed by hepatocytes from man by an active carrier-mediated transport system which is Na+-, energy- and pH-dependent and has properties very similar to those of the carnitine transporter OCTN2.

Muscle microdialysis has been used to determine the unbound concentrations of the fluoroquinolones, pazufloxacin and ofloxacin, in tissue interstitial fluids (Cisf,u) of rats under steady state conditions. Cisf,u was estimated from the concentration in dialysate and the in-vitro permeability rate constant by the extrapolation method based on the clearance concept. Paper-disks were inserted under the abdominal skin of rats, and the drug concentrations in the fluids penetrating into the disks (Cdisk) were measured and compared with Cisf,u. The Cisf,u of pazufloxacin and ofloxacin in muscle were close to their unbound concentrations in the venous plasma; these were 75.3% and 77.1%, respectively, of the total concentrations in plasma at the steady state. The Cdisk of pazufloxacin and ofloxacin were also close to their Cisf,u. These results indicate that the unbound concentrations of the fluoroquinolones in the tissue interstitial fluids were the same as those in the venous plasma. The disk insertion technique seems to be useful for evaluating drug concentrations in tissue interstitial fluid.

Although recent evidence suggests that certain beta-lactam antibiotics are absorbed via a specific transport mechanism, its nature is unclear. To confirm whether peptide transport in the rat can be largely ascribed to the intestinal oligopeptide transporter PepT1, the transporter has been functionally characterized and its significance in the intestinal absorption of beta-lactam antibiotics was evaluated. For evaluation of transport activity complementary RNA (cRNA) of rat PepT1 was synthesized in-vitro and expressed in Xenopus laevis oocytes. cRNA induced uptake of several beta-lactam antibiotics and the dipeptide [14C]glycylsarcosine; this was specifically inhibited by various dipeptides and tripeptides but not by their constituent amino acids or by tetra- or pentapeptides. The transport activity of PepT1 for beta-lactam antibiotics correlated well with their in-vivo intestinal transport and absorption. Furthermore, mutual inhibitory effects on uptake were observed between glyclsarcosine and beta-lactam antibiotics. Hybrid depletion of the functional expression of rat PepT1 in oocytes injected with rat intestinal epithelial total mRNA was studied using an antisense oligonucleotide corresponding to the 5'-coding region of PepT1. In oocytes injected with rat mRNA pre-hybridized with the antisense oligonucleotide against rat PepT1, the uptake of [14C]glycylsarcosine was almost completely abolished, whereas its uptake was not influenced by a sense oligonucleotide for the same region of PepT1. Similarly, the uptake of beta-lactam antibiotics was also reduced by the antisense oligonucleotide against rat PepT1. These results demonstrate that the intestinal proton-coupled oligopeptide transporter PepT1 plays a predominant role in the carrier-mediated intestinal absorption of beta-lactam antibiotics and native oligopeptides in the rat.

Intestinal brush-border membrane transport of monocarboxylic acids was investigated by using rabbit intestinal brush-border membrane vesicles (BBMVs) and isolated intestinal tissues mounted on Ussing-type chambers. [3H]Mevalonic acid uptake by BBMVs showed an overshoot phenomenon in the presence of an inwardly directed proton gradient, but not in the presence of an inwardly directed sodium gradient or an outwardly directed HCO3- or chloride gradient. Initial uptake of mevalonic acid was saturable in the presence of a proton gradient. Uptake of [3H]mevalonic acid was inhibited by various monocarboxylic acids, including acetic acid, benzoic acid, lactic acid, nicotinic acid, pravastatin, salicylic acid and valproic acid, but not by dicarboxylic acid or amino acids. Acetic acid, which is transported by both anion antiport and proton-coupled transport systems, induced serosal bicarbonate-dependent alkalinization in the mucosal-side bathing solution of rabbit jejunal tissues, when examined in Ussing-type chambers. Pravastatin, which is a structural analogue of mevalonic acid and is absorbed via proton-coupled transport like mevalonic acid, did not. The result demonstrates that acetic acid is transported by the bicarbonate-dependent anion antiport system, whereas pravastatin is not. So, it is suggested that monocarboxylic acids are transported by at least two independent transporters, namely, a proton-coupled transporter for most monocarboxylic acids, including mevalonic acid, pravastatin and acetic acid, and an anion antiporter for acetic acid, but not for mevalonic acid or pravastatin. Activation of anion antiporter can induce HCO3- secretion in intact intestine.

Because the permeability of the blood-brain barrier to lipid microspheres (LMs) has not hitherto been demonstrated, blood-brain-barrier permeability to LM containing the prostaglandin I2 analogue clinprost has been evaluated for an in-vitro system of primary cultured monolayers of bovine brain capillary endothelial cells (BCECs), by a capillary depletion study in rats and by an in-situ brain perfusion study in normal and 4-vessel-occluded fore brain ischaemic rats. Although energy-dependency was not observed in [3H]clinprost uptake by BCECs, in accordance with results for simple diffusional transport, uptake of [3H]clinprost contained in lipid microspheres (denoted [3H]clinprost(LM)) was significantly inhibited by the endocytosis inhibitor, dansylcadaverine. The transport of LM into BCECs by endocytosis was also confirmed by fluorescence microscopy and flow-cytometric analysis using LM labelled with a fluorescent probe, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil). The absolute uptake of Dil(LM) by BCECs, measured by HPLC, was, however, almost 1/10 that of [3H]clinprost(LM), results which suggest the superiority of simple diffusion of clinprost over endocytosis of its LM form in the uptake of clinprost(LM) by BCECs. In the capillary-depletion study with rat-brain-perfused [3H]clinprost(LM) from the internal carotid artery, the parenchyma apparent distribution volume was about 45 times larger than that of the capillary, showing that [3H]clinprost(LM) was transported through the blood-brain barrier into the brain. The permeability coefficients of [3H]clinprost and [3H]clinprost(LM) determined by insitu brain perfusion in normal rats were considerably higher than those of the active metabolite [3H]isocarbacyclin and its LM form. In addition, the Blood-brain-barrier permeabilities to [3H]clinprost, [3H]isocarbacyclin and their LM forms in ischaemic rats were almost identical to those in normal rats. It was concluded that clinprost(LM) was transported through the blood-brain barrier by endocytosis of LM, simple diffusion of clinprost released from LM, and transport of isocarbacyclin generated by hydrolysis of clinprost. The blood-brain-barrier permeability of clinprost(LM) is not reduced in ischaemic conditions, because the simple diffusion of clinprost released from LM contributed mainly to clinprost(LM) transport.

Because the significance of P-glycoprotein in the in-vivo secretion of beta-blockers in intestinal epithelial cells is unclear, the secretory mechanism for beta-blockers and other drugs has been evaluated. Uptake of the beta-blockers acebutolol, celiprolol, nadolol and timolol, and the antiarrhythmic agent, quinidine by the multidrug-resistant leukaemic cell line variant K562/ADM was significantly lower than that by drug-sensitive K562 cells, suggesting that these beta-blockers are transported by P-glycoprotein out of cells. The reduced uptake of acebutolol by the drug-resistant K562/ADM cells was reversed by treating the cells with anti-P-glycoprotein monoclonal antibody, MRK16, whereas no such alteration in uptake was observed for drug-sensitive K562 cells. Acebutolol uptake by K562/ADM cells was, moreover, markedly enhanced, in a concentration-dependent manner, in the presence of the specific P-glycoprotein inhibitors, MS-209 and cyclosporin. Caco-2 cells were used for evaluation of the role of P-glycoprotein in intestinal permeability to drugs in-vitro. Basolateral-to-apical transport of acebutolol was twice that in the reverse direction. A similar polarized flux was also observed in the transport of vinblastine, but not in that of acetamide or mannitol. When in-vivo intestinal absorption was evaluated by the rat jejunal loop method, with simultaneous intravenous administration of a P-glycoprotein inhibitor, cyclosporin, intestinal absorption of both acebutolol and vinblastine increased 2.6- and 2.2-fold, respectively, but no such enhancement was observed in the absorption of acetamide. The effect of cyclosporin on the intestinal absorption of several drugs was further examined, and the extent of the contribution of P-glycoprotein as an absorption barrier to those drugs was evaluated. ATP depletion by occlusion of the superior mesenteric artery resulted in a clear increase in epithelial permeability to vinblastine, but not to 3-O-methylglucose or acetamide, indicating that vinblastine is secreted by ATP-dependent P-glycoprotein into the lumen. These findings demonstrate that P-glycoprotein plays a role as an absorption barrier by transporting several drugs from intestinal cells into the lumen.

In order to determine whether the vitamin nicotinic acid is absorbed via an anion antiporter, intestinal epithelial cell membrane transport mechanisms for nicotinic acid were characterized using isolated rabbit jejunal brush-border membrane vesicles. The uptake of nicotinic acid by the membrane vesicles showed an overshoot phenomenon in the presence of an outwardly directed bicarbonate gradient or an inwardly directed proton gradient and the uptakes were two times and six times greater, respectively, than that in the absence of any ion gradient. The bicarbonate-dependent initial uptake of nicotinic acid was increased at acidic pH, showing pH-dependent transport activity. An inhibitor of anion transport, 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid, specifically reduced bicarbonate-dependent transport of nicotinic acid. The initial uptakes of nicotinic acid via the anion antiporter and the proton cotransporter were specifically inhibited by monocarboxylic acids such as acetic acid, benzoic acid, D- and L-lactic acid, pravastatin and valproic acid, but not by di- or tricarboxylic acids, bile acids or amino acids. Nicotinic acid uptake activity was, furthermore, expressed in a Xenopus laevis oocyte system after injection of messenger RNA (mRNA) derived from rabbit intestinal epithelial cells. These observations demonstrate that nicotinic acid is absorbed by two independent active transport mechanisms from small intestine, i.e. a proton cotransporter and an anion antiporter. The pH-dependence observed in the intestinal absorption of nicotinic acid might, therefore, be ascribed partly to pH-sensitive and partly to carrier-mediated transport mechanisms in the brush-border membrane.

The transport of monocarboxylic acid drugs such as salicylic acid was examined in the human colon adenocarcinoma cell line, Caco-2 cells that possess intestinal epithelia-like properties. [14C]Salicylic acid transport was pH-dependent and appeared to follow the pH-partition hypothesis. However, 10 mM unlabelled salicylic acid significantly reduced the permeability coefficient of [14C]salicylic acid. Kinetic analysis of the concentration dependence of the permeation rate of salicylic acid across Caco-2 cells showed both saturable (Kt = 5.28 +/- 0.72 mM Jmax = 36.6 +/- 3.54 nmol min-1 (mg protein)-1) and nonsaturable (kd = 0.37 +/- 0.08 microL min-1 (mg protein)-1) processes. The permeation rate of [14C]salicylic acid was competitively inhibited by both acetic acid and benzoic acid, which were demonstrated in our previous studies to be transported in the carrier-mediated-transport mechanism which is responsible for monocarboxylic acids. Furthermore, certain monocarboxylic acids significantly inhibited [14C]salicylic acid transport, whereas salicylamide and dicarboxylic acids such as succinic acid did not. From these results, it was concluded that the transcellular transport of [14C]salicylic acid across Caco-2 cells is by the pH-dependent and carrier-mediated transport mechanism specific for monocarboxylic acids.

Intestinal absorption of the orally active cephalosporin, cefdinir, was investigated using brush-border membrane vesicles prepared from rabbit small intestine. The initial uptake of cefdinir was pH-dependent, with increased uptake at acidic pH, and was not influenced by either sodium gradient or membrane potential difference. Cefdinir uptake was saturable with an apparent Michaelis constant of 8.1 mM. Initial uptake of cefdinir was inhibited by dipeptides (glycyl-L-proline and glycylsarcosine), beta-lactam antibiotics (cephradine, cefixime and penicillin V), and monocarboxylic acids (acetic acid and L-lactic acid), whereas the uptake of cephradine and cefixime was not inhibited by monocarboxylic acids. Cefdinir significantly inhibited the initial uptake of cephradine, cefixime and [3H]acetic acid. From these results, it was suggested that cefdinir was transported across brush-border membranes by both dipeptide and monocarboxylic acid carriers.

The mechanism of intestinal uptake of cefixime, a new oral cephalosporin antibiotic, has been examined using the everted jejunum of rats. The initial uptake rates were apparently pH-dependent with the maximum rate at pH 5.0 and a 3-fold reduction at pH 7.0. The uptake at pH 5.0 followed mixed-type kinetics involving saturable and non-saturable processes in a manner similar to that for several amino-beta-lactam antibiotics. Cefixime uptake was inhibited significantly by 20 mM permeants such as cyclacillin, cephradine, benzylpenicillin, propicillin, glycyl-L-proline and glycyl-glycine. Replacement of Na+ in the medium with choline produced a slight but significant inhibition of cefixime uptake. In spite of the absence of significant inhibition by the amino acids glycine and proline, the dipeptide, glycyl-L-proline in Na+-free medium showed a marked inhibitory effect. The inhibition kinetics of cefixime uptake by glycyl-L-proline and cyclacillin were consistent with competitive-type inhibition. This study provides the first evidence of saturable intestinal uptake of a cephem antibiotic without an alpha-amino group in the side chain, suggesting transport through the dipeptide carrier system(s).

Stereospecific absorption and degradation of two stereoisomers about the alpha-amino group at the 7-position of cephalexin (CEX) have been investigated in the rat intestine. The L-isomer (L-CEX) was not found to be present either in serum or urine after oral administration, but the D-isomer (D-CEX) was well absorbed. In contrast to the saturable uptake of D-CEX (Kt = 10.54 +/- 1.73 mM, pH = 6.0) by the in-vitro everted intestinal sac, no appreciable uptake of L-CEX was observed. However, L-CEX competitively inhibited the uptake of D-CEX by the in-vitro everted intestine and the inhibitory constant (Ki) of L-CEX was determined to be 0.67 +/- 0.09 mM. L-CEX was rapidly degraded in-vitro in the intestinal tissue homogenate, serum and urine, while there was no appreciable degradation of D-CEX. Analysis of the major metabolite of L-CEX by high-performance liquid chromatography identified it as 7-aminodeacetoxycephalosporanic acid (7-ADCA). Furthermore, 7-ADCA was detected in serum after oral administration of L-CEX, indicating significant absorption of L-CEX as well as D-CEX. The results obtained suggest that both L- and D-CEX can be absorbed through the intestinal brush-border membrane via the same mechanism, most likely through the dipeptide transport system, and that the affinity of L-CEX to the carrier system is higher than that of D-CEX.(ABSTRACT TRUNCATED AT 250 WORDS)

The transport mechanism of benzylpenicillin was studied in freshly prepared rat hepatocytes. The initial uptake rate followed both saturable and unsaturable transport processes. The Arrhenius plot of the initial uptake rate gave an activation energy of 16.8 kcal mol-1 (69 kJ mol-1). The benzylpenicillin uptake by hepatocytes was significantly inhibited by antimycin A, sodium cyanide, rotenone, 2,4-dinitrophenol, phenoxymethylpenicillin, probenecid and taurocholic acid. No significant inhibition was observed by acetylaminohippuric acid and several kinds of amino acids and dipeptides. The present study provides the first evidence for the existence of a carrier-mediated and energy-dependent transport system of benzylpenicillin in the liver.