Hitoshi Sezaki

Optimized conditions for prediction of intestinal drug permeability using Caco-2 cells

The effects of various experimental conditions on in vitro drug permeability to Caco-2 monolayers were investigated to determine the optimized conditions for the prediction of intestinal drug absorption. Concerning the pH of the transport medium in the Caco-2 study, two different pH values, 6.0 and 7.4, were tested for the apical medium with the pH of the basolateral medium fixed to 7.4. The change in the apical pH showed pronounced effects on the permeability of both passively and actively transported drugs. It was found that the transport study under the condition of an apical pH value of 6.0 showed a better prediction of in vivo drug absorption in human. The appropriate conditions for determining the permeability of poorly soluble drugs were also examined. First, the effects of bile acids, surfactant and some agents used for solubilizing drugs on the permeability and transepithelial electrical resistance (TEER) of Caco-2 monolayers were investigated. Taurocholic and cholic acid showed no effects on the permeability of 3H-Dexamethasone (DEX) and TEER at 10 mM concentration, suggesting the possibility of use in the Caco-2 study. Polyethyleneglycol-400 and dimethylsulfoxide reduced the permeability of DEX concentration dependently, whereas ethanol induced no significant changes in the permeability. Furthermore, it was demonstrated that the addition of plasma protein (bovine serum albumin) to the basolateral medium apparently facilitated the transport of poorly soluble drugs with high lipophilicity across Caco-2 monolayers. These findings clearly suggest the importance of considering the physiological conditions of in vivo drug absorption in optimizing the in vitro experimental conditions for transport study using Caco-2 cells, in order to obtain a satisfactory in vitro-in vivo correlation.

Analysis of Drug Permeation Across Caco-2 Monolayer: Implication for Predicting In Vivo Drug Absorption

AbstractPurpose. The aim of the present work is to characterize in vitro drug permeation processes across Caco-2 monolayer and to identify the advantages of this cultured cell system in predicting in vivo drug absorption after oral administration.nMethods. The passive permeability of various drugs through Caco-2 monolayer was measured using Ussing-type chambers and compared with that of the isolated rat jejunum and colon. The in vivo drug permeability to the intestinal membrane was estimated by means of an intestinal perfusion study using the rat jejunum.nResults. In Caco-2 monolayer, drug permeability increased with increasing drug lipophilicity and showed a good linear relationship with the in vivo permeability. In contrast, in the isolated jejunum and colon, the permeability of high lipophilic drugs was almost constant and, propranolol, a drug with the highest lipophilicity, hardly passed through the jejunal membrane in vitro. As a result, there was no significant relationship between in vitro and in vivo drug permeability in rat jejunum. However, the amount of drugs accumulated in the jejunal mucosa increased with increasing drug lipophilicity even under the in vitro condition.nConclusions. The permeation and the accumulation studies suggested that the rate-limiting process of in vitro permeation of lipophilic drugs through the intestinal membrane differs from that of in vivo drug absorption. On the other hand, drug permeation through Caco-2 monolayer, which consists of an epithelial cell layer and a supporting filter, is essentially the same process as that of in vivo drug absorption. We concluded that the simple monolayer structure of a cultured cell system provides a distinct advantage in predicting in vivo drug absorption.

In vitro system to evaluate oral absorption of poorly water-soluble drugs: Simultaneous analysis on dissolution and permeation of drugs

AbstractPurpose. The aim of the present work was to develop a new in vitro system to evaluate oral absorption of poorly water-soluble drugs by utilizing Caco-2 monolayers.nMethods. Caco-2 monolayer was mounted between side-by-side chambers, which enabled the simultaneous assay of dissolution and permeation of drugs (dissolution/permeation system; D/P system). Apical and basal sides of the chamber were filled with buffer solutions. Drugs were applied to the apical side as powder, suspension, or solution, and then, the permeated amounts into the basal side were monitored for 2 h. At the same time, dissolved amounts of drugs at the apical side were detected. The amount of drug applied to the D/P system was based on its in vivo clinical dose.nResults. Sodium taurocholate (5 mM, apical side) and bovine serum albumin (4.5% w/v, basal side) increased the permeated amount of poorly water-soluble drugs. Both additives were considered to be effective at mimicking in vivo conditions of intestinal drug absorption. From the correlation between the permeated amount of 13 drugs (% dose/2 h) in the D/P system and their percentage dose absorbed in humans in vivo, this system was found to be useful in evaluating oral absorption of poorly water-soluble drugs.nConclusions. With attempts made to mimic the physiologic conditions of the human GI tract, in vivo oral absorption of drugs was quantitatively assessed in the D/P system in vitro. This system is quite useful to predict the oral absorption of poorly water-soluble drugs after administration as solid dosage forms.

Direct Drug Transport from the Rat Nasal Cavity to the Cerebrospinal Fluid: the Relation to the Molecular Weight of Drugs

To clarify the relationship between the direct transport from the rat nasal cavity to the cerebrospinal fluid (CSF) and the molecular weight of the drug, the transport of fluorescein isothiocyanate‐labelled dextran (FD) with various molecular weights was investigated. FDs (average molecular weights 4400 (FD4); 9400 (FD10); 18 900 (FD20); 40 500 Da (FD40)) were administered nasally or intravenously to rats, and the concentrations in the plasma and the CSF were measured and compared.

Synthesis and Pharmacokinetics of a New Liver-Specific Carrier, Glycosylated Carboxymethyl-Dextran, and Its Application to Drug Targeting

To develop a new carrier system for hepatic targeting, carboxymethyl-dextran (CMD) was modified with galactose and mannose residues (Gal-CMD, Man-CMD), and their disposition characteristics were studied in mice using 14C-labeled dextran. At a dose of 1 mg/kg, i.v.-injected Gal-CMD and Man-CMD rapidly accumulated in the liver parenchymal and nonparenchymal cells, respectively, because of their preferential uptake via carbohydrate receptors in these cells. Pharmacokinetic analysis revealed that their uptake rates were sufficiently large for selective drug targeting. Targeting of cytosine β-D-arabinoside (araC) was studied using Gal-CMD as a specific carrier to the hepatocytes. From the conjugate of araC with Gal-CMD, araC was released with a half-life of 36 hr in phosphate buffer (pH 7.4) and 23 hr in plasma. An in vivo biodistribution study demonstrated a disposition profile of the conjugated araC similar to that of the carrier, and selective delivery to hepatocytes of up to 80% of the dose was achieved. These findings suggest that glycosylated CMDs are carriers with a high affinity to liver parenchymal or nonparenchymal cells without any affinity to other tissues.

Direct drug transport from the rat nasal cavity to the cerebrospinal fluid: the relation to the dissociation of the drug

Abstract— We aimed to clarify the relationship between drug dissociation (sulphisomidine) and its direct transport from the nasal cavity to the cerebrospinal fluid (CSF). Rat nasal cavities were perfused in a single pass system with buffers (pH 5·5, 6·5, 7·4, 8·7 and 9·4). Plasma and CSF were collected and the concentration of sulphisomidine was measured. Nasal clearance increased with the increase in the un‐ionized fraction of the drug. The ratio of the drug concentration in CSF to that in the nasal perfusion fluid (the index of the degree of the drug transport from the nasal cavity to CSF), was changed in accordance with the un‐ionized fraction of drug. These results show that both the nasal absorption and the drug transport conform to the pH partition theory.

Transnasal delivery of 5-fluorouracil to the brain in the rat.

The purpose of this research is to clarify the feasibility and to determine the extent of transnasal drug delivery to the brain through the cerebrospinal fluid (CSF) in the rat, using 3H-5-fluorouracil (5FU) as a model drug. It was confirmed first that the concentration of 5FU in the CSF was significantly higher following nasal administration compared with intravenous injection, indicating direct transport of 5FU from the nasal cavity to the CSF. Concentration-time profiles of 5FU in the plasma and in the cerebral cortex were determined following intravenous infusion, nasal instillation and nasal perfusion. In order to evaluate the extent of drug transport from the nasal cavity to the cerebral cortex by way of the CSF, the apparent brain uptake clearances were calculated. The uptake clearance following nasal perfusion (8.65 microl/min/g tissue) was significantly large (p < 0.001) in comparison with that following intravenous infusion (6.20 microl/min/g tissue), while that following nasal instillation (6.94 microl/min/g tissue) was not. Consequently, significant amount of 5FU is transported from the nasal cavity to the brain through the CSF and thus, the delivery of the hydrophilic drug to the brain is augmented by nasal drug application.

Improvement of the Pulmonary Absorption of (Asu1,7)-Eel Calcitonin by Various Protease Inhibitors in Rats

The effects of protease inhibitors, Na-glycocholate, bacitracin, bestatin, nafamostat mesilate and soybean trypsin inhibitor (STI) on the pulmonary absorption of (Asu1,7)-eel calcitonin (ECT, molecular weight 3363) were investigated in rats. The pulmonary absorption of ECT was estimated by measuring its hypocalcemic effect. When ECT alone was administered into the lung, the pharmacological availability of ECT was 2.7%. Co-administration with STI or bestatin did not change the pharmacological effect of ECT. However, Na-glycocholate, bacitracin and nafamostat mesilate caused a significant hypocalcemic effect following the pulmonary absorption of ECT and a maximal effect was noted in the presence of 20 mM bacitracin, approaching the effect after intravenous administration of ECT. Bacitracin and Na-glycocholate reduced the degradation of 111In-ECT in rat lung homogenate. Therefore, protease inhibitors effectively improved the pulmonary absorption of ECT.

Controlled Biodistribution of Highly Lipophilic Drugs with Various Parenteral Formulations

Lipid carrier systems are considered effective for targeting highly lipophilic drugs, but little systematic information about the effect of the physicochemical and pharmaceutical characteristics of drugs and formulations on their performance has been obtained. 3H-Retinoic acid and 14C-cholesteryl oleate with different lipophilicities (log PCoct = 6.6 and 18, respectively) were selected as model drugs and the potential of formulations such as oil in water (o/w) emulsion, micellar solution, and liposomes for controlling their biodistribution was demonstrated. After intravenous injection in mice, 3H-retinoic acid showed similar disposition profiles irrespective of formulation type, suggesting its rapid dissociation from carriers. 14C-Cholesteryl oleate with extremely high lipophilicity revealed widely varied disposition profiles reflecting the distribution patterns of carriers: micellar solution and liposomes showed large AUC values and low hepatic clearances, while the use of emulsion as a carrier resulted in rapid clearance from blood circulation into the liver. The results suggested that these formulations can be used as targeting carriers for lipophilic drugs which, however, should have a sufficiently high lipophilicity of about log PCoct 9-16.

Control of In Vivo Fate of Albumin Derivatives Utilizing Combined Chemical Modification

Three types of bovine serum albumin (BSA) derivatives such as lactosylated BSA (LBSA), mannosylated BSA (Man-BSA), and cationized BSA (cBSA) were synthesized and their hepatic disposition characteristics in mice were evaluated by pharmacokinetic analysis. At lower doses (< or = 1 mg/kg), LBSA and Man-BSA were very rapidly eliminated from the blood circulation due to uptake by parenchymal and nonparenchymal cells of the liver, respectively, via receptor-mediated endocytosis (Nishikawa et al., 1992; Nishida et al., 1991a, b). These uptake processes were nonlinear and the apparent hepatic uptake clearances (CLliver) were decreased at administered doses higher than 1 mg/kg, e.g. 10, 20, and 100 mg/kg. The liver accumulation of cBSA was also nonlinear, but its binding and/or uptake capacity in the liver was larger than those of LBSA and Man-BSA; i.e., CLliver decreased at doses higher than 20 mg/kg. In the next step, we modified these BSA derivatives by attaching polyethylene glycol (PEG), a modifier known to reduce the hepatic uptake and increase plasma retention, to achieve precise control of the in vivo disposition characteristics of BSA derivatives. By conjugation with PEG having a molecular weight of 10 kDa, the CLliver values of LBSA, Man-BSA, and cBSA were decreasing to one-seventh, one-fortyfifth, and one-onehundredthirtieth, respectively. However, liver accumulation of PEG modified LBSA and Man-BSA at 24 h after i.v. injection was not significantly different from unmodified BSA derivatives. These results suggest that it is possible to control the hepatic uptake of protein drugs by a combination of introduction of charge or sugar moieties and PEG conjugation.