Shoji Awazu

In vitro and in vivo evaluation of the tissue-to-blood partition coefficient for physiological pharmacokinetic models.

An important parameter used in physiologically based pharmacokinetic models is the partition coefficient (Kp), which is defined as the ratio of tissue drug concentration to the concentration of drug in the emergent venous blood of the tissue. Since Kp is governed by reversible binding to protein and other constituents in blood and tissue, an attempt was made here to estimate the Kp values for a model drug ethoxybenzamide (EB) by means of in vitrobinding studies and to compare these Kp values to those obtained from in vivokinetic parameters observed following the administration of EB by two different routes, i.e., i.v. bolus injection and constant rate infusion. The Kp values obtained by using these three different methods were in reasonably good agreement suggesting that binding data obtained in vitrocan successfully be used to estimate in vivodistribution.

Enhancement of Colonic Drug Absorption by the Transcellular Permeation Route

The effects of sodium caprate and sodium caprylate on transcellular permeation routes were examined in rats. The release of membrane phospholipids was significantly increased only by caprate, while protein release did not change from the control in the presence of caprate or caprylate, indicating that the extent of membrane disruption was insufficient to account for enhanced permeation. Using brush border membrane (BBM) vesicles prepared from colon, with their protein and lipid component labeled by fluorescent probes, the perturbing actions of caprate and caprylate toward the membrane were examined by fluorescence polarization. Caprate interacted with membrane protein and lipids, and caprylate mainly with protein, causing perturbation to the membrane. The release of 5(6)-carboxyfluorescein previously included in BBM vesicles was increased by caprate but not by caprylate. These results suggest that caprate enhances permeability via the transcellular route through membrane perturbation.

Physiological pharmacokinetics of ethoxybenzamide based on biochemical data obtained in vitro as well as on physiological data.

Ethoxybenzamide (EB) concentrations in plasma and various tissues were simulated using a physiological pharmacokinetic model. The biochemical parameters, such as plasma and tissue binding constants and Michaelis-Menten constants for EB deethylation, which were needed for these simulations, were, however, obtained from in vitro data. The simulations predicted well the observed data in plasma and various tissues of the rat. Furthermore, animal scale-up predicted reasonably well the concentrations of EB in plasma and various tissues of the rabbit from data gathered in rats.

Comparative physiologically based pharmacokinetics of hexobarbital, phenobarbital, and thiopental in the rat

A physiologically based pharmacokinetic model, which is an extension of the Bischoff-Dedrick multiorgan model, was developed to describe the kinetics of barbiturates (hexobarbital, phenobarbital, and thiopental) in the rat. The model is composed of 11 organ or tissue compartments. The brain compartment was featured as a nonflow-limited organ for some low lipid soluble barbiturates. Michaelis-Menten constants for drug metabolism (Km, Vmaxwere determined from in vitroexperiments using liver microsomes. Binding of drugs to plasma and tissue proteins was measured in vitrousing an equilibrium dialysis method. Distribution of drugs to red blood cells was measured in vitrowith thiopental exhibiting a concentration dependent distribution. Penetration rates of the barbiturates into the brain were predicted on the basis of their lipid solubilities. A set of mass balance equations included terms for the inflow and outflow of drug carried by the perfusing blood, drug metabolism, protein binding, and penetration rate into the brain as well as blood flow rate and tissue mass. Solution of the system of equations yielded the time courses of drugs in each organ. However, predicted time courses of drugs in plasma and brain were not in good agreement with those observed. Therefore, the tissue to plasma distribution ratios evaluated from in vivoexperiments were substituted for the in vitrovalues, resulting in fairly good agreement between predicted and observed values.

Intestinal active absorption of sugar-conjugated compounds by glucose transport system: implication of improvement of poorly absorbable drugs.

The intestinal absorption of glucose- and galactose-conjugated compounds was studied in the everted sac of the rat small intestine. The absorption clearance of p-nitrophenyl beta-D-glucopyranoside (p-NPglc) at 250 microM in the mucosal side (4.45 +/- 0.34 microL/min/cm, mean +/- SE, N = 4), calculated by dividing the absorption rate by the drug concentration, was significantly decreased (0.476 +/- 0.036 microL/min/cm) in the presence of 1 mM phloridzin, an inhibitor of glucose transport, and in the absence of Na+, a cosubstrate of the glucose transport carrier (0.424 +/- 0.018 microL/min/cm). The absorption clearance of p-NPglc was decreased as its concentration increased. In the same experiment, the absorption clearance of p-nitrophenyl beta-D-galactopyranoside (1.99 +/- 0.23 microL/min/cm) was also significantly decreased in the presence of phloridzin and in the absence of Na+. However, the absorption clearance of p-nitrophenyl beta-D-mannopyranoside (0.811 +/- 0.013 microL/min/cm) was low and not significantly decreased in the presence of phloridzin (P greater than 0.1). Furthermore, the absorption clearance of beta-naphthyl beta-D-glucopyranoside and beta-naphthyl beta-D-galactopyranoside was also significantly decreased in the presence of phloridzin (P less than 0.001). These results indicated that the glucose and galactose moieties provided these compounds with a new route by way of the glucose transport carrier for intestinal absorption.

Physiological mechanism for enhancement of paracellular drug transport.

We examined the action mechanisms of enhancers that improve paracellular drug transport. For sodium caprate (C10), the increase in the intracellular calcium level was considered to induce the contraction of calmodulin-dependent actin filaments, followed by dilation of the paracellular pathway. Although decanoylcarnitine (DC) also increased the intracellular calcium level, the action was independent of calmodulin and thus, the action mechanism of acylcarnitines was considered to differ from that of C10. Other acylcarnitines, lauroylcarnitine (LC) and palmitoylcarnitine (PC) and organic acids, tartaric acid (TA) and citric acid (CA) decreased the intracellular ATP level and the intracellular pH. From these results, it was considered that one of the action mechanism of acylcarnitines and organic acids is that the intracellular acidosis increases the calcium level through the decrease in ATP levels, followed by opening the tight junction. Membrane dysfunction which was expected from the above mechanism was assessed by the transport function of electrolytes. Membrane conductance, which was increased by C10, LC and PC, returned to the control value during a 3- to 6-h recovery period. On the other hand, Cl(-) ion secretion, which was obtained from short-circuit current (I(sc)), was decreased by these enhancers, but was normalized by C10 but not by LC and PC. Accordingly, C10 can be considered a safer enhancer than acylcarnitines.

The β-anomeric and glucose preferences of glucose transport carrier for intestinal active absorption of monosaccharide conjugates

The anomeric preference for intestinal absorption of glucosides and galactosides (1- and 2-naphthyl glycosides) was studied by the everted rat intestine method. After the absorption of beta-glucoside and beta- and alpha-galactosides, the glycosides itself appeared on the serosal side, whereas after the alpha-glucoside absorption, the glucoside itself was not detected on the serosal side, but a large amount of aglycone appeared instead. This indicates an alpha-anomeric preference of desglucosylation through the intestinal membrane. A significant decrease of the total (glycoside + aglycone + glucuronide metabolites) amount transported to the serosal side in the absence of Na+, a cosubstrate of the glucose transport carrier (GTC), was observed in alpha- and beta-glucosides and beta-galactoside, but not in alpha-galactoside. This indicates the poor contribution of GTC to the alpha-galactoside absorption. The Na(+)-dependent absorption of the glycosides by the GTC were beta-glucoside > alpha-glucoside > beta-galactoside (for 2-naphthyl glycosides), and beta-galactoside > alpha-galactoside (for 1-naphthhyl glycosides). These results and those of a previous study led to the conclusion that the intestinal glucose transport carrier prefers beta-anomer to alpha-anomer, and glucose to galactose for monosaccharide conjugates absorption.

Role of Paracellular Pathway in Nonelectrolyte Permeation Across Rat Colon Epithelium Enhanced by Sodium Caprate and Sodium Caprylate

The enhancing effects of 0.25% sodium caprate (C10) and sodium caprylate (C8) on the paracellular permeation of seven water-soluble nonelectrolytes (inulin, polyethylene glycol 900, mannitol, erythritol, glycerol, thiourea, and urea) across the isolated rat colonic epithelium were examined using the Ussing-type chamber technique. The paracellular changes were also measured by impedance analysis. In both the presence and the absence of enhancers, the permeation clearances (Pm) for inulin (12–15 Å in molecular radius) to erythritol (3.2 Å) increased linearly with the increase in their free diffusion coefficients (Dfr), showing the existence of a paracellular shunt pathway unrestricted to any molecular size. Glycerol (2.9 Å), thiourea (2.6 Å), and urea (2.3 Å) had higher clearances than the expected linear values, showing the existence of a restricted paracellular or transcellular pathway. Both C10 and C8 increased the permeabilities in the two pathways, but C10 was more effective than C8. The increase in the permeabilities via the shunt pathway caused by the enhancers was greater than that via the restricted pathway, and thus, the two-phase pattern in the relationship of Pm and Dfr was similar to that in the absence of enhancers. The transcellular permeabilities for urea and thiourea, which were obtained from the efflux experiments, were increased by the enhancers. However, the relative increase caused by C10 was smaller than that of the paracellular-restricted permeabilities. The paracellular changes probably were due to the increase in pore area per unit diffusive path length. A decrease in the resistance of the intercellular junctions involving a simultaneous increase of membrane capacitance was observed in the presence of C10, corresponding to an increase of pore area per unit path length. The effect of C10 on the paracellular permeability was reversible, and the junctional resistance, membrane capacitance, and Pm of mannitol returned to the control level following the removal of C10.

Correlation between in vitro and in vivo drug metabolism rate: oxidation of ethoxybenzamide in rat.

In vitroand in vivocorrelations of the microsomal oxidation of drugs were examined, using ethoxybenzamide as a model drug. Ethoxybenzamide disappearance time course from rat plasma in vivowas analyzed by a two-compartment model assuming a Michaelis-Menten type elimination process. Ethoxybenazmide oxidation in vitrowas measured by the appearance rate of salicylamide in rat liver microsomal suspension. Parameters obtained were Vmax=3.46 and 3.77 μmoles/min/kg body weight and Km=0.378 and 0.192mM, in vitroand in vivo,respectively.

Absorption-Enhancing Effects of Sodium Caprate and Palmitoyl Carnitine in Rat and Human Colons

We examined the enhancing action of sodiumcaprate and palmitoylcarnitine on the permeability offluorescein isothiocyanate dextran 4000 as aparacellular permeant compound in isolated rat and humancolon samples using the Ussing-type chamber method.In the absence of an enhancer, the permeation clearanceof fluorescein isothiocyanate dextran 4000 was notsignificantly different in the rat and human colons, but the electric membrane resistance wassmaller in the rat colon than in the human colon. Sodiumcaprate and palmitoylcarnitine increased permeationclearance and decreased electric membrane resistance in both types of colonic membrane, showing thatthe rat colon can be used as a model of the human colonfor studies of enhancer effects. A calmodulin antagonistsignificantly inhibited the action of sodium caprate in both colonic membranes. However, ittended to promote the effects of palmitoylcarnitine onpermeation clearance and electric membrane resistance.These results suggest that sodium caprate induces the contraction of the perijunctionalactomyosin ring to widen the tight junction and that themechanism of palmitoylcarnitine is different from thatof sodium caprate in the human colon, as reportedpreviously for Caco-2 cell monolayers.