Saturable Process Involved in Active Efflux of Vincristine as a Mechanism of Multidrug Resistance in P388 Leukemia Cells

Kinetic analysis of vincristine (VCR) efflux in multidrug-resistant and parental P388 leukemia cells was performed to investigate the difference in activity between the two cell lines. Efflux velocities of VCR were directly determined from the slope of the initial release of drug induced by resuspending the preloaded cells in VCR-free medium, representing unidirectional efflux from intracellular free or loosely bound drug pools. Further, the equilibrium binding of VCR to whole-cell homogenates was analyzed by ultrafiltration to estimate intracellular unbound drug concentrations. A two-site binding model was found to fit the data best for both cell lines, and depletion of ATP by the addition of apyrase decreased binding. The binding parameters were similar between the two cell lines. A Hofstee plot of efflux demonstrated the existence of both linear and saturable transport of VCR in both cell lines. The greater maximum velocity observed with VCR efflux in the resistant cells suggests that an increased number of transporters causes greater activity of this process in the resistant cells.     

Estimating Human Oral Fraction Dose Absorbed: A Correlation Using Rat Intestinal Membrane Permeability for Passive and Carrier-Mediated Compounds

Based on a simple tube model for drug absorption, the key parameters controlling drug absorption are shown to be the dimensionless effective permeability, P _eff ^*, and the Graetz number, Gz, when metabolism or solubility/dissolution is not rate controlling. Estimating the Graetz number in humans and assuming that P _aq ^* is not rate controlling gives the following equation for fraction dose absorbed: F = 1– e ^{–2 P*w}. The correlation between fraction dose absorbed in humans and P _w ^* determined from steady-state perfused rat intestinal segments gives an excellent correlation. It is of particular significance that the correlation includes drugs that are absorbed by passive and carrier-mediated processes. This indicates that P _w ^* is one of the key variables controlling oral drug absorption and that the correlation may be useful for estimating oral drug absorption in humans regardless of the mechanism of absorption.     

Analysis of Hepatic Transport of Cefpiramide in Rats with Obstructive Jaundice by Using Isolated Hepatocytes

The mechanism of the diminished biliary clearance of cefpiramide (CPM) in rats with obstructive jaundice (OJ) was investigated by using isolated hepatocytes. The kinetics of CPM uptake by hepatocytes isolated from normal rats and rats with OJ could be explained by the combination of saturable carrier-mediated and nonsaturable first-order rate processes. The maximum uptake rate (V _max) of the carrier-mediated process was significantly decreased in OJ, compared with normal hepatocytes, while the Michaelis constant (K _m) and the first-order rate constant (k _d) were not significantly different. This result indicated that the number of CPM transport carriers was decreased in OJ hepatocytes. Further, no CPM uptake occurred from the serum of OJ rats into normal hepatocytes. Partial recovery of CPM uptake after treatment of OJ serum with activated charcoal suggested the accumulation of inhibitors of CPM uptake in OJ serum.     

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.     

Transport of Pregabalin in Rat Intestine and Caco-2 Monolayers

Purpose. The purpose of this study was to determine if the intestinal transport of pregabalin (isobutyl --aminobutyric acid, isobutyl GAB A), a new anticonvulsant drug, was mediated by amino acid carriers with affinity for large neutral amino acids (LNAA). Methods. Pregabalin transport was studied in rat intestine and Caco-2 monolayers. An in vitro Ussing/diffusion chamber model and an in situ single-pass perfusion model were used to study rat intestinal transport. An in vitro diffusion chamber model was used to evaluate Caco-2 transport. Results. In rat ileum, pregabalin transport was saturable and inhibited by substrates of intestinal LNAA carriers including neurontin (gabapentin), phenylalanine, and proline. Weak substrates of intestinal LNAA carriers (-alanine, --aminobutyric acid, and methyl aminoisobutyric acid) did not significantly change pregabalin transport. In Caco-2 mono-layers that showed a high capacity for phenylalanine transport, pregabalin transport was concentration- and direction-independent and equivalent in magnitude to the paracellular marker, mannitol. The in vitro and in situ rat ileal permeabilities of the LNAA carrier-mediated compounds neurontin, pregabalin, and phenylalanine correlated well with the corresponding in vivo human oral absorption. Conclusions. The transport of pregabalin was mediated by LNAA carriers in rat ileum but not in Caco-2 monolayers. Caco-2 was not an appropriate model for evaluating the in vivo human oral absorption of pregabalin and neurontin.     

Carrier mediated transport of choline in rat lens

Choline accumulation was studied in rat lenses incubated in TC-199 medium containing radiolabeled choline. Choline entered the lens and was rapidly phosphorylated. Phosphorylcholine did not readily escape the lens and continued to accumulate throughout 24 hr of incubation. Accumulation of choline displayed saturation kinetics and this saturability appeared to be a property of transport rather than a reflection of the properties of choline kinase. Countertransport of labeled choline from lenses preloaded with radiolabeled choline indicates that choline transport in rat lens is carrier mediated. The existence of a choline carrier would also be consistent with the kinetic data. Ethanolamine competed for the choline carrier, however a component of ethanolamine uptake was non-saturable at concentrations of ethanolamine or choline up to 5 mm. Choline and ethanolamine appeared to be phosphorylated by separate kinases in lens.     

Solute Absorption from the Airways of the Isolated Rat Lung. IV. Mechanisms of Absorption of Fluorophore-Labeled Poly-α,β-[N(2-Hydroxyethyl)-DL-Aspartamide]

The pulmonary absorption kinetics of a single molecular weight distribution (MWD) of fluorophore-labeled poly-,-[N(2-hydroxyethyl)-DL-aspartamide] (F-PHEA), a hydrophilic and biocompatible synthetic polypeptide, were studied in the isolated, perfused rat lung (iprl) as functions of administered polymer concentration, dose, vehicle, and presence and absence of fluorophore. The MWD was characterized before and after absorption by measurement of weight- and number-averaged molecular weights (M _wand M _n, respectively) using high-performance gel-permeation chromatography. Values for M _w and M _n were 8.6 and 5.3 kD before, and 6.7 and 4.7 kD after, absorption into the perfusate; there was no significant metabolism and the MWD of the absorbed polymer was independent of both dose and sampling time over a 3-hr period. F-PHEA failed to show any evidence of aggregation in solution or changes in dose distribution within the airways as functions of increasing polymer concentration and dose. A concentration ranging study indicated the presence of a saturable, carrier-mediated transport process for F-PHEA with a maximum absorption rate, V _max, of approximately 180 µg or 0.027 µmol/hr. Coadministration of fluorophore-free PHEA was capable of depressing the absorption of F-PHEA. The transport process for F-PHEA appeared to have a molecular weight limit of about 7 kD for this hydrophilic polymer.     

Blood–brain barrier endogenous transporters as therapeutic targets: a new model for small molecule CNS drug discovery

Introduction: The blood–brain barrier (BBB) limits the uptake of most drugs by brain, and the traditional approach to the BBB problem is the use of medicinal chemistry to increase drug lipid solubility, and increase lipid-mediated transport across the BBB. This review advocates a new model to CNS drug discovery of BBB-penetrating small molecules, whereby drug candidates are screened for carrier-mediated transport (CMT) across the BBB.

Areas covered: CMT systems are expressed by genes within the Solute Carrier (SLC) Transporter Gene Family, which now totals > 400 transporter genes. Emphasis is placed on reconciliation of the substrate transporter profile (STP) of BBB transport in vivo with the STP of the cloned SLC transporter in vitro. This reconciliation is crucial to the identification, from sometimes a large number of candidates, of the respective SLC transporter that is responsible for BBB transport in vivo for a given class of nutrients.

Expert opinion: Dual track screening of a small molecule library for drugs that have the dual properties of affinity for a neural cell drug receptor target, and affinity for a BBB CMT transporter target, can lead to a revolution in how small molecule drugs are identified in CNS drug discovery programs.     

Transcellular transport of aconitine across human intestinal Caco-2 cells

Aconitine (AC) is a highly toxic compound present in plants of the genus Aconitum. The transcellular transport mechanism of AC was investigated using Caco-2 cells. The flux of AC was time- and concentration-dependent in both apical-to-basolateral and the reverse direction. The efflux of AC was more than two-fold that in the opposite direction. The influx of AC was temperature-, pH- and Na⁺-dependent. Glucose markedly decreased the absorption of AC. However, the efflux of AC was temperature- and pH-dependent, but Na⁺-independent. Cyclosporin A and verapamil, both inhibitors of P-glycoprotein (P-gp), significantly decreased the efflux of AC. In addition, MK-571, an inhibitor of multidrug resistance-associated protein 2 (MRP2), exhibited the same trend but to a lesser extent. These results indicate that both the influx and efflux of AC across Caco-2 monolayers were through an active process. A pH-dependent carrier-mediated transport system was the major absorption mechanism and a sodium-dependent glucose transporter may be involved. The active efflux of AC across Caco-2 cells was mediated mainly by ABC-transporter P-gp. It is involved in reducing the toxicity of AC to organisms and is the major reasons for the poor absorption of AC in vivo.     

Determination of Transport Rates for Arginine and Acetaminophen in Rabbit Intestinal Tissues in Vitro

The in vitro Ussing technique was employed to examine transport rates for acetaminophen and arginine across rabbit intestinal tissues. Mannitol and transepithelial conductance were used to monitor the integrity of rabbit intestinal tissues and the basal and stimulated short-circuit current were measured to assess functional viability. Transepithelial transport of acetaminophen, arginine, and mannitol was determined in rabbit jejunum, ileum, and distal colon. Transepithelial transport of arginine in the ileum and jejunum was composed of both passive (nonsaturable) (P _m = 0.06) and saturable components (K _T = 0.6-0.7 mM; J _max = 0.3-0.4 µmol/hr · cm²). The saturable component of arginine fluxes was abolished by pretreatment of the tissue with serosal ouabain (0.1 mM). In the distal colon, both unidirectional arginine fluxes were nonsaturable. In the segments examined, both unidirectional fluxes of acetaminophen were nonsaturable over the concentration range from 0.1 to 30 mM. These results provide values for maximal permeabilities attained by molecules traversing both the cellular and the paracellular pathways and, by comparison to their in vivo bioavailabilities, provide selection criteria for evaluating drug candidates for oral activity.