RESEARCH ARTICLES
A Systematic Examination of the In Vitro Ussing Chamber and the In Situ Single-Pass Perfusion Model Systems in Rat Ileum Permeation of Model Solutes

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ABSTRACT:

In situ and in vitro intestinal absorption in the rat ileum was systematically studied and mechanistically quantified in terms of permeability coefficients (P) of a series of [3H]steroids as model transcellular permeants, [3H]taurocholate utilizing the active membrane transport systems to define the aqueous boundary layer (ABL), and [14C]urea and [14C]mannitol as pore-hindered paracellular diffusants. In situ single-pass perfusion experiments were performed in isolated ileal segments and blood samples were collected from the cannulated mesenteric vein. For the in vitro experiments, an excised, serosal and muscular layer-removed, ileal tissue was mounted in the Ussing chamber diffusion cells. In situ and in vitro P values versus logarithm of the partition coefficient in n-octanol/water (log K) of the steroids were characterized by a sigmoidal-shaped curve in which plateau values were attained for the highly lipophilic steroids with log K ≳ 2.5. The in situ and in vitro transport barriers in series were viewed as ABL/mucosal epithelium and ABL/mucosal epithelium/submucosal tissue, respectively. Within this framework and the use of experimental strategies and theoretical reasoning, the transport barriers of the steroids were quantitatively delineated and the rate-determining barriers identified. In the plateau region, the analyses indicate that the in situ absorption of the lipophilic steroids was essentially ABL controlled, whereas the in vitro absorption was about equally controlled by diffusion across the ABL and submucosal tissue. The in situ and in vitro pore radii of the paracellular route were 7.2 and 9.2 Å, respectively, and the difference was likely the result of perturbation of the tight junctions during the in vitro preparation of the ileal tissue.

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INTRODUCTION

The in vitro Ussing chamber system using an animal mucosal membrane mounted in the diffusion cells to evaluate the absorption characteristics of new drugs at the membrane level has been gaining in popularity in recent years.1., 2., 3., 4. In situ methods in animal models have also been used to determine absorption mechanisms.5., 6., 7. It has been reported that the in vitro permeability of drugs is lower than that in vivo. Lennernas et al.1 suggested that the differences in quantitative

Chemicals and Reagents

For the in vitro Ussing chamber system, a trace amount of radiolabeled permeants was added to pH 7.4 Krebs-bicarbonate Ringer's solution (KBR). The KBR had the following composition (mM): 108.0 NaCl, 4.7 KCl, 1.8 Na2HPO4, 0.4 NaH2PO4, 15.0 NaHCO3, 1.2 MgSO4, 1.25 CaCl2, 11.5 D-glucose. For the in situ system, modified Krebs-Ringer's solution (MKBR) and pH 7.4 phosphate buffer solution were used as the medium. The phosphate buffer solution had the following composition (mM): 57 NaH2PO4, 79 Na2SO4.

Absorption of Steroid Series

In situ absorption studies were systematically performed with a series of steroids varying in lipophilicity, but similar in molecular size. The isoosmotic perfusion solution contained [3H]steroid, phenol red, and MKBR at pH 7.4. Figure 2 shows the fraction of [3H]ED remaining in the perfused intestinal segment, C(ℓ)/C(0), with time and concurrent appearance in blood with time. The condition of steady-state was achieved in 20 min. Thereafter, C(ℓ)/C(0) reached a plateau and the absorption rate in

SIGNIFICANCE

A systematic and mechanistic approach has been applied to understand and quantify the rate-determining factors and barriers in the in situ and in vitro intestinal absorption of a common series of steroids in the rat ileum. Experimental strategies were gainfully employed to define the mass transfer resistances of the ABL, mucosal epithelium, and the submucosal tissue. TC absorption in the ileum had a key role in establishing the permeability of the ABL. Whereas both the in situ and in vitro

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