Targeted PEG-based bioconjugates enhance the cellular uptake and transport of a HIV-1 TAT nonapeptide.

We previously described the enhanced cell uptake and transport of R.I-K(biotin)-Tat9, a large ( approximately 1500 Da) peptidic inhibitor of HIV-1 Tat protein, via SMVT, the intestinal biotin transporter. The aim of the present study was to investigate the feasibility of targeting biotinylated PEG-based conjugates to SMVT in order to enhance cell uptake and transport of Tat9. The 29 kDa peptide-loaded bioconjugate (PEG:(R.I-Cys-K(biotin)-Tat9)8) used in these studies contained eight copies of R.I-K(biotin)-Tat9 appended to PEG by means of a cysteine linkage. The absorptive transport of biotin-PEG-3400 (0.6-100 microM) and the bioconjugate (0.1-30 microM) was studied using Caco-2 cell monolayers. Inhibition of biotin-PEG-3400 by positive controls (biotin, biocytin, and desthiobiotin) was also determined. Uptake of these two compounds was also determined in CHO cells transfected with human SMVT (CHO/hSMVT) and control cells (CHO/pSPORT) over the concentration ranges of 0.05-12.5 microM and 0.003-30 microM, respectively. Nonbiotinylated forms of these two compounds, PEG-3350 and PEG:(R.I-Cys-K-Tat9)8, were used in the control studies. Biotin-PEG-3400 transport was found to be concentration-dependent and saturable in Caco-2 cells (K(m)=6.61 microM) and CHO/hSMVT cells (K(m)=1.26 microM). Transport/uptake was significantly inhibited by positive control substrates of SMVT. PEG:(R.I-Cys-K(biotin)Tat9)8 also showed saturable transport kinetics in Caco-2 cells (K(m)=6.13 microM) and CHO/hSMVT cells (K(m)=8.19 microM). Maximal uptake in molar equivalents of R.I-Cys-K(biotin)Tat9 was 5.7 times greater using the conjugate versus the biotinylated peptide alone. Transport of the nonbiotinylated forms was significantly lower (P<0.001) in all cases. The present results demonstrate that biotin-PEG-3400 and PEG:(R.I-Cys-K(biotin)Tat9)8 interact with human SMVT to enhance the cellular uptake and transport of these larger molecules and that targeted bioconjugates may have potential for enhancing the cellular uptake and transport of small peptide therapeutic agents.     

Transport of amino acid-based prodrugs by the Na+- and Cl(-) -coupled amino acid transporter ATB0,+ and expression of the transporter in tissues amenable for drug delivery

We evaluated the potential of the Na(+)- and Cl(-)-coupled amino acid transporter ATB(0,+) as a delivery system for amino acid-based prodrugs. Immunofluorescence analysis indicated that ATB(0,+) is expressed abundantly on the luminal surface of cells lining the lumen of the large intestine and the airways of the lung and in various ocular tissues, including the conjunctival epithelium, the tissues easily amenable for drug delivery. We screened a variety of beta-carboxyl derivatives of aspartate and gamma-carboxyl derivatives of glutamate as potential substrates for this transporter using heterologous expression systems. In mammalian cells expressing the cloned ATB(0,+), several of the aspartate and glutamate derivatives inhibited glycine transport via ATB(0,+). Direct evidence for ATB(0,+)-mediated transport of these derivatives was obtained in Xenopus laevis oocytes using electrophysiological methods. Exposure of oocytes, which express ATB(0,+) heterologously, to aspartate beta-benzyl ester as a model derivative induced inward currents in a Na(+)- and Cl(-)-dependent manner with a Na(+)/Cl(-)/aspartate beta-benzyl ester stoichiometry of 2:1:1. ATB(0,+) transported not only the beta-carboxyl derivatives of aspartate and the gamma-carboxyl derivatives of glutamate but also valacyclovir, which is an alpha-carboxyl ester of acyclovir with valine. The transport of valacyclovir via ATB(0,+) was demonstrable in both heterologous expression systems. This process was dependent on Na(+) and Cl(-). The ability of ATB(0,+) to transport valacyclovir was comparable with that of the peptide transporter PEPT1. These findings suggest that ATB(0,+) has significant potential as a delivery system for amino acid-based drugs and prodrugs.     

Stimulus-dependent modulation of [(3)H]norepinephrine release from rat neocortical slices by gabapentin and pregabalin

Gabapentin (GBP; Neurontin) has proven efficacy in several neurological and psychiatric disorders yet its mechanism of action remains elusive. This drug, and the related compounds pregabalin [PGB; CI-1008, S-(+)-3-isobutylgaba] and its enantiomer R-(-)-3-isobutylgaba, were tested in an in vitro superfusion model of stimulation-evoked neurotransmitter release using rat neocortical slices prelabeled with [(3)H]norepinephrine ([(3)H]NE). The variables addressed were stimulus type (i.e., electrical, K(+), veratridine) and intensity, concentration dependence, onset and reversibility of action, and commonality of mechanism. Both GBP and PGB inhibited electrically and K(+)-evoked [(3)H]NE release, but not that induced by veratridine. Inhibition by these drugs was most pronounced with the K(+) stimulus, allowing determination of concentration-effect relationships (viz., 25 mM K(+) stimulus: GBP IC(50) = 8.9 microM, PGB IC(50) = 11.8 microM). R-(-)-3-Isobutylgaba was less effective than PGB to decrease stimulation-evoked [(3)H]NE release. Other experiments with GBP demonstrated the dependence of [(3)H]NE release inhibition on optimal stimulus intensity. The inhibitory effect of GBP increased with longer slice exposure time before stimulation, and reversed upon washout. Combination experiments with GBP and PGB indicated a similar mechanism of action to inhibit K(+)-evoked [(3)H]NE release. GBP and PGB are concluded to act in a comparable, if not identical, manner to preferentially attenuate [(3)H]NE release evoked by stimuli effecting mild and prolonged depolarizations. This type of modulation of neurotransmitter release may be integral to the clinical pharmacology of these drugs.     

Transport of ochratoxin A by renal multispecific organic anion transporter 1.

In the present study, we investigated the transport of ochratoxin A (OTA) by kidney-specific organic anion transporter 1 (OAT1). When expressed in Xenopus laevis oocytes, OAT1 mediated sodium-independent uptake of OTA (Km = 2.1 microM). Piroxicam, which has been shown to prevent the nephrotoxicity of OTA, inhibited OAT1-mediated uptake of OTA. By contrast, another protective compound, aspartame, did not. Using a cell line derived from the mouse kidney terminal proximal tubule (S3) transfected with OAT1 cDNA, we investigated the transport of OTA and also its effect on cell proliferation and cell viability. S3 cells expressing OAT1 mediated the saturable transport of OTA (Km = 0.57 microM). Cell proliferation was suppressed in S3 cells expressing OAT1 when exposed to 2 and 10 microM OTA. This suppression was rescued by the coaddition of 1 mM p-aminohippurate in the media. The present study indicates that OTA is transported by OAT1 and that the accumulation of OTA via OAT1 in proximal tubular cells is the primary event in the development of OTA nephrotoxicity.     

Interactions of a nonpeptidic drug, valacyclovir, with the human intestinal peptide transporter (hPEPT1) expressed in a mammalian cell line

The results of previous work performed in our laboratory using an in situ perfusion technique in rats and rabbit apical brush border membrane vesicles have suggested that the intestinal uptake of valacyclovir (VACV) appears to be mediated by multiple membrane transporters. Using these techniques, it is difficult to characterize the transport kinetics of VACV with each individual transporter in the presence of multiple known or unknown transporters. The purpose of this study was to characterize the interaction of VACV and the human intestinal peptide transporter using Chinese hamster ovary (CHO) cells that overexpress the human intestinal peptide transporter (hPEPT1) gene. VACV uptake was significantly greater in CHO cells transfected with hPEPT1 than in cells transfected with only the vector, pcDNA3. The optimum pH for VACV uptake was determined to occur at pH 7.5. Proton cotransport was not observed in hPEPT1/CHO cells, consistent with previously observed results in tissues and Caco-2 cells. VACV uptake was concentration dependent and saturable with a Michaelis-Menten constant and maximum velocity of 1.64 +/- 0.06 mM and 23.34 +/- 0.36 nmol/mg protein/5 min, respectively. A very similar Km value was obtained in hPEPT1/CHO cells and in rat and rabbit tissues and Caco-2 cells, suggesting that hPEPT1 dominates the intestinal transport properties of VACV in vitro. VACV uptake was markedly inhibited by various dipeptides and beta-lactam antibiotics, and Ki values of 12.8 +/- 2.7 and 9.1 +/- 1.2 mM were obtained for Gly-Sar and cefadroxil at pH 7.5, respectively. The present results demonstrate that VACV is a substrate for the human intestinal peptide transporter in hPEPT1/CHO cells and that although transport is pH dependent, proton cotransport is not apparent. Also, the results demonstrate that the hPEPT1/CHO cell system has use in investigating the transport kinetics of drugs with the human intestinal peptide transporter hPEPT1; however, the extrapolation of these transport properties to the in vivo situation requires further investigation.     

Hepatic uptake of the magnetic resonance imaging contrast agent gadoxetate by the organic anion transporting polypeptide Oatp1.

Gadoxetate is a new hepatobiliary magnetic resonance imaging contrast agent. It is specifically taken up by hepatocytes, and its uptake can be inhibited by the coadministration of bromosulfophthalein, suggesting an involvement of one or several of the cloned organic anion transporting polypeptides Oatp1, Oatp2, and/or OATP. In this study, we demonstrated saturable uptake of gadoxetate by Oatp1 cRNA-injected Xenopus laevis oocytes (Km approximately 3.3 mM). In contrast, gadoxetate was not taken up by Oatp2 or OATP cRNA-injected oocytes. Oatp1-mediated gadoxetate uptake (100 microM) could be inhibited by 10 microM bromosulfophthalein (45%), 200 microM taurocholate (92%), 100 microM rifamycin SV (97%), and 100 microM rifampicin (51%). These results show that gadoxetate is a low-affinity substrate of Oatp1. Oatp1-mediated gadoxetate transport demonstrated a similar apparent Km value and cis-inhibition pattern as previously determined in rats in vivo, indicating that Oatp1 is significantly involved in gadoxetate uptake into rat liver.     

Na+ - and Cl- -coupled active transport of nitric oxide synthase inhibitors via amino acid transport system B(0,+)

Nitric oxide synthase (NOS) inhibitors have therapeutic potential in the management of numerous conditions in which NO overproduction plays a critical role. Identification of transport systems in the intestine that can mediate the uptake of NOS inhibitors is important to assess the oral bioavailability and therapeutic efficacy of these potential drugs. Here, we have cloned the Na+ - and Cl- -coupled amino acid transport system B(0,+) (ATB(0,+)) from the mouse colon and investigated its ability to transport NOS inhibitors. When expressed in mammalian cells, ATB(0,+) can transport a variety of zwitterionic and cationic amino acids in a Na+ - and Cl- -coupled manner. Each of the NOS inhibitors tested compete with glycine for uptake through this transport system. Furthermore, using a tritiated analog of the NOS inhibitor N(G)-nitro-L-arginine, we showed that Na+ - and Cl- -coupled transport occurs via ATB(0,+). We then studied transport of a wide variety of NOS inhibitors in Xenopus laevis oocytes expressing the cloned ATB(0,+) and found that ATB(0,+) can transport a broad range of zwitterionic or cationic NOS inhibitors. These data represent the first identification of an ion gradient-driven transport system for NOS inhibitors in the intestinal tract.     

Amperometric measurement of glutamate release modulation by gabapentin and pregabalin in rat neocortical slices: role of voltage-sensitive Ca2+ α2δ-1 subunit

Gabapentin (GBP; Neurontin) and pregabalin (PGB; Lyrica, S-(+)-3-isobutylgaba) are used clinically to treat several disorders associated with excessive or inappropriate excitability, including epilepsy; pain from diabetic neuropathy, postherpetic neuralgia, and fibromyalgia; and generalized anxiety disorder. The molecular basis for these drugs' therapeutic effects are believed to involve the interaction with the auxiliary α(2)δ subunit of voltage-sensitive Ca(2+) channel (VSCC) translating into a modulation of pathological neurotransmitter release. Glutamate as the primary excitatory neurotransmitter in the mammalian central nervous system contributes, under conditions of excessive glutamate release, to neurological and psychiatric disorders. This study used enzyme-based microelectrode arrays to directly measure extracellular glutamate release in rat neocortical slices and determine the modulation of this release by GBP and PGB. Both drugs attenuated K(+)-evoked glutamate release without affecting basal glutamate levels. PGB (0.1-100 μM) exhibited concentration-dependent inhibition of K(+)-evoked glutamate release with an IC(50) value of 5.3 μM. R-(-)-3-Isobutylgaba, the enantiomer of PGB, did not significantly reduce K(+)-evoked glutamate release. The decrease of K(+)-evoked glutamate release by PGB was blocked by the l-amino acid l-isoleucine, a potential endogenous ligand of the α(2)δ subunit. In neocortical slices from transgenic mice having a point mutation (i.e., R217A) of the α(2)δ-1 (subtype) subunit of VSCC, PGB did not affect K(+)-evoked glutamate release yet inhibited this release in wild-type mice. The results show that GBP and PGB attenuated stimulus-evoked glutamate release in rodent neocortical slices and that the α(2)δ-1 subunit of VSCC appears to mediate this effect.     

Involvement of concentrative nucleoside transporter 1 in intestinal absorption of trifluorothymidine, a novel antitumor nucleoside, in rats

ααα-Trifluorothymidine (TFT), an anticancer nucleoside analog, is a potent thymidylate synthase inhibitor. TFT exerts its antitumor activity primarily by inducing DNA fragmentation after incorporation of the triphosphate form of TFT into the DNA. Although an oral combination of TFT and a thymidine phosphorylase inhibitor has been clinically developed, there is little information regarding TFT absorption. Therefore, we investigated TFT absorption in the rat small intestine. After oral administration of TFT in rats, more than 75% of the TFT was absorbed. To identify the uptake transport system, uptake studies were conducted by using everted sacs prepared from rat small intestines. TFT uptake was saturable, significantly reduced under Na(+)-free conditions, and strongly inhibited by the addition of an endogenous pyrimidine nucleoside. From these results, we suggested the involvement of concentrative nucleoside transporters (CNTs) in TFT absorption into rat small intestine. In rat small intestines, the mRNAs coding for rat CNT1 (rCNT1) and rCNT2, but not for rCNT3, were predominantly expressed. To investigate the roles of rCNT1 and rCNT2 in TFT uptake, we conducted uptake assays by using Xenopus laevis oocytes injected with rCNT1 complementary RNA (cRNA) and rCNT2 cRNA. TFT uptake by X. laevis oocytes injected with rCNT1 cRNA, and not rCNT2 cRNA, was significantly greater than that by water-injected oocytes. In addition, in situ single-pass perfusion experiments performed using rat jejunum regions showed that thymidine, a substrate for CNT1, strongly inhibited TFT uptake. In conclusion, TFT is absorbed via rCNT1 in the intestinal lumen in rats.     

Differential substrate and inhibitory activities of ranitidine and famotidine toward human organic cation transporter 1 (hOCT1; SLC22A1), hOCT2 (SLC22A2), and hOCT3 (SLC22A3)

Human organic cation transporters (hOCTs) are expressed in organs of drug absorption and elimination and play an important role in the uptake and elimination of xenobiotics. The purpose of this study was to evaluate the substrate and inhibitory activity of the H2-receptor antagonists ranitidine and famotidine toward hOCTs and to determine the hOCT isoforms involved in the absorption and elimination of these compounds in humans. Inhibition and substrate specificity of hOCT1, hOCT2, and hOCT3 for ranitidine and famotidine were elucidated in cRNA-injected Xenopus laevis oocytes. Ranitidine and famotidine exhibited similarly potent inhibition of [3H]1-methyl-4-phenyl pyridinium uptake into hOCT1-expressing (IC50= 33 and 28 microM, respectively) and hOCT2-expressing oocytes (IC50= 76 and 114 microM, respectively). Famotidine exhibited potent inhibition of hOCT3; in contrast, ranitidine was a moderately weak inhibitor (IC50= 6.7 and 290 microM, respectively). [3H]Ranitidine uptake was stimulated by hOCT1 (Km= 70 +/- 9 microM) and to a much smaller extent by hOCT2. No stimulation of [3H]ranitidine uptake was observed in hOCT3-expressing oocytes. trans-Stimulation and electrophysiology studies suggested that famotidine also is an hOCT1 substrate and exhibits poor or no substrate activity toward hOCT2 and hOCT3. Thus, hOCT1, which is expressed in the intestine and liver, is likely to play a major role in the intestinal absorption and hepatic disposition of ranitidine and famotidine in humans, whereas hOCT2, the major isoform present in the kidney, may play only a minor role in their renal elimination. Famotidine seems to be one of the most potent inhibitors of hOCT3 yet identified.     

Mechanism of intestinal absorption of an orally active beta-lactam prodrug: uptake and transport of carindacillin in Caco-2 cells.

Absorption characteristics of carindacillin (CIPC) were investigated using Caco-2 cells, and the results were compared with those of its parent drug, carbenicillin (CBPC). Uptake of CBPC was not affected by the metabolic inhibitor or the change in extracellular pH. CBPC appeared to be taken up into Caco-2 cells by passive diffusion. In contrast, the uptake of CIPC was greater at lower extracellular pH and was inhibited in the presence of carbonyl cyanide p-(trifluoromethoxy)phenyl hydrazone, a protonophore. Also, transport of CIPC through Caco-2 cell monolayer was energy and temperature dependent. Moreover, the uptake and transport of CIPC were significantly inhibited in the presence of various monocarboxylic acids, which are the substrates of the monocarboxylic acid transport system(s), whereas the substrates of the oligopeptide transporter had no effect on the uptake or transport of CIPC. These results suggested that the absorption of CIPC may be mediated by the monocarboxylic acid transport system(s), not by the oligopeptide transporter. Furthermore, the uptake and transport of CIPC were approximately 40-fold greater than those of CBPC. Therefore, it is likely that the participation of a carrier-mediated transport in the absorption of CIPC may significantly contribute to the improved absorption of the prodrug over the parent drug.     

Transport of the natural sweetener stevioside and its aglycone steviol by human organic anion transporter (hOAT1; SLC22A6) and hOAT3 (SLC22A8)

The natural sweetening agent stevioside and its aglycone metabolite, steviol, have been shown to inhibit transepithelial transport of para-aminohippurate (PAH) in isolated rabbit renal proximal tubules by interfering with basolateral entry. The aim of the present study was to determine which of the cloned basolateral organic anion transporters were involved in the renal transport of stevioside and steviol. This question was addressed in Xenopus laevis oocytes expressing human organic anion transporter 1 (hOAT1), 3 (hOAT3), and winter flounder OAT (fOat1). The parent compound, stevioside, had no inhibitory effect on either PAH (hOAT1) or ES (estrone sulfate; hOAT3) uptake. In contrast, steviol showed significant, dose-dependent inhibition of PAH and ES uptake in hOAT1- or hOAT3-expressing oocytes, respectively. The IC(50) of steviol for hOAT1-mediated PAH transport was 11.1 microM compared with 62.6 microM for hOAT3-mediated ES uptake. The Michaelis-Menten inhibition constants (K(i)) for steviol transport mediated by hOAT1 and hOAT3 were 2.0 +/- 0.3 and 5.4 +/- 2.0 microM, respectively. Trans-stimulation of PAH efflux by steviol was assessed to determine whether steviol itself was transported by hOAT1 or hOAT3. A low concentration of 1 microM steviol increased the efflux of [(3)H]PAH (trans-stimulated) via both hOAT1 and hOAT3. In addition, it was shown by electrophysiology that steviol entry induced inward current in fOat1-expressing oocytes. In conclusion, stevioside had no interaction with either hOAT1 or hOAT3, whereas hOAT1, hOAT3, and fOat1 were all shown to be capable of steviol transport and thus, can play a role in its renal transport and excretion.     

Cotransport of macrolide and fluoroquinolones, a beneficial interaction reversing P-glycoprotein efflux

The purpose of this study was to determine the interactions of erythromycin and various fluoroquinolones with P-glycoprotein (P-gp) and in turn assess their effects on transport kinetics across a model cell monolayer. MDCKII-MDRI cells were selected as a model monolayer to evaluate the effects of various fluoroquinolones, ie, norfloxacin, lomefloxacin, ofloxacin, enoxacin, grepafloxacin, levofloxacin, and sparfloxacin on the P-gp-mediated efflux of H-cyclosporine (CsA) and C-erythromycin. IC50 values associated with grepafloxacin-, levofloxacin-, and sparfloxacin-mediated inhibition of P-gp were calculated across Caco-2 cells with erythromycin as the model P-gp substrate. Transport of erythromycin was then studied with P-gp saturable concentrations of fluoroquinolones. Western blot analysis was performed on Caco-2 cells to confirm P-gp expression. Only grepafloxacin elevated the uptake of H-CsA across the MDCKII-MDRI cell monolayer, whereas norfloxacin, lomefloxacin, ofloxacin, and enoxacin did not exert any effect on H-CsA uptake. Inhibition studies indicate that grepafloxacin, levofloxacin, and sparfloxacin are potent inhibitors of P-gp-mediated efflux of C-erythromycin in the MDCKII-MDRI cell monolayer. Similar studies were conducted across Caco-2 cells and IC50 values were calculated. Inhibitory potency of sparfloxacin (IC50 = 607.6 microM) exceeded that of levofloxacin (IC50 = 1644 microM) and grepafloxacin (IC50 = 2266 microM). Permeability ratio (BL-AP/AP-BL) of C-erythromycin was found to be 8.67, which was reduced to 1.18, 1.83, and 1.39 in the presence of grepafloxacin (1 mmol/L), levofloxacin (5 mmol/L), and sparfloxacin (1 mmol/L), respectively. Log partition coefficient of grepafloxacin (1.58), levofloxacin (0.553), and sparfloxacin (0.45) were correlated with the inhibition of P-gp. Western blot analysis indicated the expression of P-gp in Caco-2 cells. Fluoroquinolones like grepafloxacin, levofloxacin, and especially sparfloxacin significantly inhibit the efflux of erythromycin, which can modulate oral absorption and disposition of macrolide drugs when administered concomitantly.     

Transport of angiotensin-converting enzyme inhibitors by H+/peptide transporters revisited

Angiotensin-converting enzyme (ACE) inhibitors are often regarded as substrates for the H+/peptide transporters (PEPT)1 and PEPT2. Even though the conclusions drawn from published data are quite inconsistent, in most review articles PEPT1 is claimed to mediate the intestinal absorption of ACE inhibitors and thus to determine their oral availability. We systematically investigated the interaction of a series of ACE inhibitors with PEPT1 and PEPT2. First, we studied the effect of 14 ACE inhibitors including new drugs on the uptake of the dipeptide [14C]glycylsarcosine into human intestinal Caco-2 cells constitutively expressing PEPT1 and rat renal SKPT cells expressing PEPT2. In a second approach, the interaction of ACE inhibitors with heterologously expressed human PEPT1 and PEPT2 was determined. In both assay systems, zofenopril and fosinopril were found to have very high affinity for binding to peptide transporters. Medium to low affinity for transporter interaction was found for benazepril, quinapril, trandolapril, spirapril, cilazapril, ramipril, moexipril, quinaprilat, and perindopril. For enalapril, lisinopril, and captopril, very weak affinity or lack of interaction was found. Transport currents of PEPT1 and PEPT2 expressed in Xenopus laevis oocytes were recorded by the two-electrode voltage-clamp technique. Statistically significant, but very low currents were only observed for lisinopril, enalapril, quinapril, and benazepril at PEPT1 and for spirapril at PEPT2. For the other ACE inhibitors, electrogenic transport activity was extremely low or not measurable at all. The present results suggest that peptide transporters do not control intestinal absorption and renal reabsorption of ACE inhibitors.     

Delineation of human peptide transporter 1 (hPepT1)-mediated uptake and transport of substrates with varying transporter affinities utilizing stably transfected hPepT1/Madin-Darby canine kidney clones and Caco-2 cells

In the present investigation, the uptake and transport kinetics of valacyclovir (VACV), 5-aminolevulinic acid (5-ALA), and benzylpenicillin (BENZ) were studied in stably transfected Madin-Darby canine kidney (MDCK)/human peptide transporter 1 (hPepT1)-V5&His clonal cell lines expressing varying levels of epitope-tagged hPepT1 protein (low, medium, and high expression) and in Caco-2 cells to delineate hPepT1-mediated transport kinetics. These compounds were selected due to the fact that they are known PepT1 substrates, yet also have affinity for other transporters. Caco-2 cells, traditionally used for studying peptide-based drug transport, were included for comparison purposes. The time, pH, sodium, and concentration dependence of cellular uptake and permeability were measured using mock, clonal hPepT1-MDCK, and Caco-2 cells. A pH-dependent effect was observed in the hPepT1-expressing clones and Caco-2 cells, with an increase of 1.96-, 1.84-, and 2.05-fold for VACV, 5-ALA, and BENZ uptake, respectively, at pH 6 versus 7.4 in the high-expressing hPepT1 cells. BENZ uptake was significantly decreased in Caco-2 and MDCK cells in Na(+)-depleted buffer, whereas VACV uptake only decreased in Caco-2 cells. Concentration-dependent uptake studies in the mock-corrected hPepT1-MDCK and Caco-2 cells demonstrated hPepT1 affinity ranking of VACV > 5-ALA > BENZ. The apical-to-basal apparent permeability coefficient (P(app)) values of VACV, 5-ALA, and BENZ in mock-corrected hPepT1-MDCK cells showed solely hPepT1-mediated transport in contrast to Caco-2 cells. Lower K(m) values and higher P(app) in Caco-2 cells compared with hPepT1-MDCK cells suggested the involvement of multiple transporters in Caco-2 cells. Thus, hPepT1-MDCK cells corrected for endogenous transporter expression may be a more appropriate model for screening compounds for their affinity to hPepT1.     

Involvement of rat and human organic anion transporter 3 in the renal tubular secretion of topotecan [(S)-9-dimethylaminomethyl-10-hydroxy-camptothecin hydrochloride

Topotecan [(S)-9-dimethylaminomethyl-10-hydroxy-camptothecin hydrochloride] is primarily excreted into urine in humans, with approximately 49% of the dose recovered as total topotecan (topotecan lactone plus topotecan hydroxyl acid form). The renal elimination of topotecan involves tubular secretion in addition to glomerular filtration, but little is known about the molecular mechanism of the renal tubular secretion. In the present study, we investigated the transport characteristics of topotecan hydroxyl acid across the renal basolateral membrane using rat kidney slices and rat or human transporter-expressing Xenopus laevis oocytes. Pravastatin and probenecid significantly inhibited the uptake of topotecan hydroxyl acid by rat kidney slices with K(i) values of 10.6 and 8.1 microM, respectively, and p-aminohippurate was weakly inhibitory at high concentrations, whereas excess tetraethylammonium had no effect. The uptake of topotecan hydroxyl acid by oocytes injected with complementary RNA of either rat or human organic anion transporter 3 (rOAT3 or hOAT3) was greater than that of water-injected oocytes. Kinetic analysis showed that the K(m) values for rOAT3 and hOAT3 were 21.9 and 56.5 microM, respectively. Neither rOAT1 nor hOAT1 stimulated topotecan hydroxyl acid transport. These results suggest that the urinary excretion of topotecan hydroxyl acid is accounted for by transport via OAT3, as well as glomerular filtration, in both rats and humans; therefore, drug-drug interactions involving OAT3 may cause a change in clearance of topotecan.     

Raltegravir is a substrate for SLC22A6: a putative mechanism for the interaction between raltegravir and tenofovir

The identification of transporters of the HIV integrase inhibitor raltegravir could be a factor in an understanding of the pharmacokinetic-pharmacodynamic relationship and reported drug interactions of raltegravir. Here we determined whether raltegravir was a substrate for ABCB1 or the influx transporters SLCO1A2, SLCO1B1, SLCO1B3, SLC22A1, SLC22A6, SLC10A1, SLC15A1, and SLC15A2. Raltegravir transport by ABCB1 was studied with CEM, CEM(VBL100), and Caco-2 cells. Transport by uptake transporters was assessed by using a Xenopus laevis oocyte expression system, peripheral blood mononuclear cells, and primary renal cells. The kinetics of raltegravir transport and competition between raltegravir and tenofovir were also investigated using SLC22A6-expressing oocytes. Raltegravir was confirmed to be an ABCB1 substrate in CEM, CEM(VBL100), and Caco-2 cells. Raltegravir was also transported by SLC22A6 and SLC15A1 in oocyte expression systems but not by other transporters studied. The K(m) and V(max) for SLC22A6 transport were 150 μM and 36 pmol/oocyte/h, respectively. Tenofovir and raltegravir competed for SLC22A6 transport in a concentration-dependent manner. Raltegravir inhibited 1 μM tenofovir with a 50% inhibitory concentration (IC(50)) of 14.0 μM, and tenofovir inhibited 1 μM raltegravir with an IC(50) of 27.3 μM. Raltegravir concentrations were not altered by transporter inhibitors in peripheral blood mononuclear cells or primary renal cells. Raltegravir is a substrate for SLC22A6 and SLC15A1 in the oocyte expression system. However, transport was limited compared to endogenous controls, and these transporters are unlikely to have a great impact on raltegravir pharmacokinetics.     

Transport characteristics of a novel peptide transporter 1 substrate, antihypotensive drug midodrine, and its amino acid derivatives

Midodrine is an oral drug for orthostatic hypotension. This drug is almost completely absorbed after oral administration and converted into its active form, 1-(2',5'-dimethoxyphenyl)-2-aminoethanol) (DMAE), by the cleavage of a glycine residue. The intestinal H+-coupled peptide transporter 1 (PEPT1) transports various peptide-like drugs and has been used as a target molecule for improving the intestinal absorption of poorly absorbed drugs through amino acid modifications. Because midodrine meets these requirements, we examined whether midodrine can be a substrate for PEPT1. The uptake of midodrine, but not DMAE, was markedly increased in PEPT1-expressing oocytes compared with water-injected oocytes. Midodrine uptake by Caco-2 cells was saturable and was inhibited by various PEPT1 substrates. Midodrine absorption from the rat intestine was very rapid and was significantly inhibited by the high-affinity PEPT1 substrate cyclacillin, assessed by the alteration of the area under the blood concentration-time curve for 30 min and the maximal concentration. Some amino acid derivatives of DMAE were transported by PEPT1, and their transport was dependent on the amino acids modified. In contrast to neutral substrates, cationic midodrine was taken up extensively at alkaline pH, and this pH profile was reproduced by a 14-state model of PEPT1, which we recently reported. These findings indicate that PEPT1 can transport midodrine and contributes to the high bioavailability of this drug and that Gly modification of DMAE is desirable for a prodrug of DMAE.     

Transport characteristics of diphenhydramine in human intestinal epithelial Caco-2 cells: contribution of pH-dependent transport system.

Transport characteristics of diphenhydramine, an antihistamine, were studied in cultured human intestinal Caco-2 cell monolayers to elucidate the mechanisms of its intestinal absorption. Diphenhydramine accumulation in the monolayers increased rapidly and was influenced by extracellular pH (pH 7.4 > 6.5 > 5.5). Diphenhydramine uptake was temperature dependent, saturable, and not potential sensitive. Kinetic analysis revealed that the apparent Km values were constant (0.8-1.0 mM) in all pH conditions tested, whereas Vmax values decreased at the lower pH. The initial uptake of diphenhydramine was competitively inhibited by another antihistamine, chlorpheniramine, with a Ki value of 1.3 mM. On the other hand, cimetidine and tetraethylammonium, typical substrates for the renal organic cation transport system, had no effect. Moreover, biological amines and neurotransmitters, such as histamine, dopamine, serotonin, and choline, also had no effect on the diphenhydramine accumulation. Finally, diphenhydramine uptake was stimulated by preloading monolayers with chlorpheniramine (trans-stimulation effect). These findings indicate that diphenhydramine transport in Caco-2 cells is mediated by a specific transport system. This pH-dependent transport system may contribute to the intestinal absorption of diphenhydramine.     

Rat multispecific organic anion transporter 1 (rOAT1) transports zidovudine, acyclovir, and other antiviral nucleoside analogs

Organic anion transporter 1 (OAT1) is a p-aminohippurate/dicarboxylate exchanger that plays a primary role in the tubular secretion of endogenous and exogenous organic anions. OAT1 is located in the basolateral membrane of the proximal tubular cells and mediates the uptake of various organic anions from the peritubular fluid. In this study, we investigated the transport of antiviral nucleoside analogs via rat OAT1 (rOAT1) using a heterologous expression system in Xenopus laevis oocytes. Oocytes injected with rOAT1 cRNA showed significantly higher uptake of zidovudine (AZT) and acyclovir (ACV) than control oocytes. rOAT1-mediated uptake of AZT and ACV was probenecid-sensitive and increased by the outwardly directed gradient of glutarate. The affinity of rOAT1 for AZT and ACV was determined to be 68 and 242 microM, respectively. Five other antiviral agents that we studied (zalcitabine, didanosine, lamivudine, stavudine, and trifluridine) were also shown to be transported by rOAT1, whereas foscarnet, a phosphate analog, was not. The aforementioned nucleoside analogs lack a typical anionic group and are not very hydrophobic. This study demonstrates extension of the substrate spectrum of rOAT1 and provides a molecular basis for the pharmacokinetics of antiviral nucleoside analogs.