Ikumi Tamai

Carrier-Mediated Intestinal Transport of Drugs

Recent advances in the field of carrier-mediated intestinal absorption of of amino acids, oligopeptides, monosaccharides, monocarboxylic acids, phosphate, bile acids and several water-soluble vitamins across brush-border and basolateral membranes are summarized. An understanding of the molecular and functional characteristics of the intestinal membrane transporters will be helpful in the utilization of these transporters for the enhanced oral delivery of poorly absorbed drugs. Some successful examples of the synthesis of prodrugs recognized by the targeted transporters are described. Functional expression of the multidrug resistance gene product, P-glycoprotein, as a primary active transporter in the intestinal brush-border membrane leads to net secretion of some drugs such as anticancer agents in the blood-to-luminal direction, serving as a secretory detoxifying mechanism and as a part of the absorption barrier in the intestine.

Sequence, tissue distribution and developmental changes in rat intestinal oligopeptide transporter

Complementary DNA clones encoding the rat PepT1 small-intestinal oligopeptide transporter were isolated from a jejunal library by cross-hybridization with a rabbit PepT1 cDNA probe. The cDNA sequence indicates that rat PepT1 is composed of 710 amino acids and shows 77% and 83% amino acid sequence identity with rabbit and human PepT1, respectively. Northern blot analysis detected rat PepT1 mRNA in the small intestine and kidney. Intestinal PepT1 mRNA levels were highest in 4-day old rats, and then decreased reaching the adult level by day 28 after birth. These results indicate that the expressions of PepT1 gene change markedly during development.

Drug delivery through the blood-brain barrier

Abstract Specific mechanisms functioning at the blood-brain barrier (BBB) for the permeation of drugs and natural compounds in the blood-to-brain and brain-to-blood directions are described. Various anionic compounds are transported by monocarboxylic acid-specific transporter(s). One such transporter is MCT1, which transports lactic acid, short-chain acids and others. As regards cationic compounds, at least two types of transporter for amines and cationic drugs appear to operate. Relatively small peptides are also transported by carrier-mediated transport mechanisms. Peptides can be delivered into the brain by absorptive-mediated endocytosis (transcytosis), which is specific to positively charged peptides or peptides into which positive charges have been introduced. For more specific delivery into brain, receptor-mediated endocytosis for peptides carrying both BBB-transporting and pharmacologically active moieties is promising. Furthermore, specific drug efflux from the brain mediated by P-glycoprotein is described. The utilization and/or avoidance of these specific mechanisms at the BBB should make it possible to enhance delivery of drugs into the brain, or to decrease the apparent permeability in order to prevent undesirable side effects in the central nervous system.

In vivo and in vitro evidence for ATP-dependency of P-glycoprotein-mediated efflux of doxorubicin at the blood-brain barrier.

We investigated the role of ATP in the active efflux of doxorubicin (DOX) mediated by P-glycoprotein (P-gp), the multidrug-resistance (MDR) gene product, at the blood-brain barrier. In transient brain ischemic rats prepared with 4-vessel occlusion of vertebral and common carotid arteries for 20 min, a procedure that depleted their brain ATP content to 3% that of normal rats, the estimated permeability coefficient of DOX was increased 17-fold (to 243 +/- 2.5 microL/min/g brain). When the ATP content recovered to a normal level by means of 30-min and 24-hr cerebral recirculation of blood, the permeability coefficient recovered to 14.0 +/- 5.0 and 18.4 +/- 2.3 microL/min/g brain (mean +/- SEM, N = 3-6), respectively, very close to the control permeability (14.3 +/- 1.5 microL/min/g brain). The uptake of DOX by primary cultured brain capillary endothelial cells expressing P-gp at the luminal membrane was increased significantly (up to 2-fold), which correlated well with the decrease of cellular ATP contents caused by treating the cells with metabolic inhibitors. Evidence for the ATP-dependent transport of DOX obtained from the present in vivo and in vitro studies strongly indicates that P-gp in the brain capillaries functions actively as an efflux pump in the physiological state, providing a major mechanism to restrict the transfer of DOX into the brain.

Proton-cotransport of pravastatin across intestinal brush-border membrane.

AbstractPurpose. 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.nMethods. 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.nResults. 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 (pHin/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 (pHin/out = 7.5/5.5), the initial uptake of pravastatin was saturable with the apparent Kt of 15.2 ± 3.2 mM and Jmax 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.nConclusions. 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.

Na+- and Cl−-Dependent transport of taurine at the blood-brain barrier

The characteristics of carrier-mediated transport of taurine at the blood-brain barrier (BBB) were studied by using primary cultured bovine brain capillary endothelial cells (BCECs), in situ brain perfusion and brain capillary depletion methods in rats. The uptake of [3H]taurine by cultured cells showed that the active transporter functions on both the luminal and antiluminal membranes of BCECs. The kinetic parameters for the saturable transport of taurine were estimated to be: for the luminal uptake, the Michaelis constant, Kt, was 12.1 +/- 0.5 microM, and the maximum uptake rate, Jmax, was 4.32 +/- 0.05 nmol/30 min/mg protein; for the antiluminal uptake, Kt was 13.6 +/- 2.4 microM and Jmax was 2.81 +/- 0.22 nmol/30 min/mg protein. The luminal and antiluminal uptakes of [3H]taurine were each dependent on both Na+ Cl-. Stoichiometric analyses suggest that two Na+ and one Cl- are associated with the luminal uptake of one taurine molecule. beta-Amino acids such as beta-alanine and hypotaurine strongly inhibited the uptake of [3H]taurine, whereas alpha- and gamma-amino acids had little or no effect. Furthermore, by in situ brain perfusion and in vivo brain capillary depletion methods, the carrier-mediated transport found by in vitro experiments was confirmed to function for the translocation of the taurine molecule from the vascular space into the brain. From these results, it was concluded that a Na+ and Cl- gradient-dependent transport (uptake) system for taurine exists in both the luminal and the antiluminal membranes of BCECs.

Carrier-mediated approaches for oral drug delivery

Abstract Intestinal brush-border and basolateral membrane transport mechanisms for monosaccharides, monocarboxylic acids, phosphate and several water-soluble vitamins are summarized and possible utilization of such transport mechanisms for the enhancement of intestinal absorption of drugs is discussed. Molecular structures and functional characteristics of hexose transporters have been well clarified. Brush-border transport of hexoses occurs via two different transporters, sodium ion-dependent SGLT1 and sodium ion-independent GLUT5, which have differential substrate specificities. Basolateral membrane transport is mediated by sodium ion-independent GLUT2 for most hexoses. Several sugar analogues are also substrates of these transporters. Monocarboxylic acids, including lactic acid and short-chain fatty acids, have been shown to be transported by proton-coupled and/or bicarbonate exchange transport mechanisms. A cDNA clone of the possible lactic acid transporter, MCT1, was obtained from rat intestine, in which transport is energized by a proton gradient. Several acidic drugs including salicylic acid and its analogues and the HMG-CoA reductase inhibitor, pravastatin, are thought to be absorbed via the monocarboxylic acid transporters. Phosphate transport across intestinal brush-border membranes is mediated by sodium ion-and pH-sensitive mechanisms. Although the molecular features of the intestinal phosphate transporters have not been fully clarified yet, the antiviral agent, foscarnet and the antibacterial agent, fosfomycin, which each has a phosphate moiety within the molecule, were suggested to be absorbed via the phosphate transporters. Carrier-mediated transport of several water-soluble vitamins, including nicotinic acid, folic acid, ascorbic acid, biotin, choline and pantothenic acid, has also been proposed. Methotrexate is absorbed via the folic acid transporter, but it is not known whether the other vitamin transporters function in the absorption of structurally analogous drugs. An understanding of the molecular and functional characteristics of the intestinal membrane transporters will be helpful in the utilization of these transporters for the enhanced oral delivery of poorly absorbable drugs.

Stereoselective and carrier-mediated transport of monocarboxylic acids across Caco-2 cells

AbstractPurpose. To characterize the transport mechanism of monocarboxylic acids across intestinal epithelial cells by examining the stereoselectivity of the transcellular transport of several chiral monocarboxylic acids.nMethods. The transport of monocarboxylic acids was examined using monolayers of human adenocarcinoma cell line, Caco-2 cells.nResults. The permeability of L-[14C]lactic acid at a tracer concentration (1 µM) exhibited pH- and concentration-dependencies and was significantly greater than that of the D-isomer. The permeabilities of both L-/ D-[14C]lactic acids involve saturable and nonsaturable processes; the saturable process showed a higher affinity and a lower capacity for L-lactic acid compared with the D-isomer, while no difference between the isomers was seen for the nonsaturable process. The transport of L-lactic acid was inhibited by chiral monocarboxylic acids such as (R)/(S)-mandelic acids and (R)/(S)-ibuprofen in a stereoselective manner. Mutually competitive inhibition was observed between L-lactic acid and (S)-mandelic acid.nConclusions. Some chiral monocarboxylic acids are transported across the intestinal epithelial cells in a stereoselective manner by the specific carrier-mediated transport mechanism.

Nicotinic acid transport mediated by pH-dependent anion antiporter and proton cotransporter in rabbit intestinal brush-border membrane

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.

Intestinal Brush‐border Membrane Transport of Monocarboxylic Acids Mediated by Proton‐coupled Transport and Anion Antiport Mechanisms

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.