Physico-chemical descriptors of drug molecules are often not adequate in predicting their oral bioavailability. In vitro methods can be useful in evaluating some of the different factors contributing to bioavailability. While physical parameters such as drug solubility may effect oral bioavailability, in most cases, the major determining factors are likely to be metabolism, and absorption at the intestinal level. Metabolism may be preabsorptive, as with peptides, or during absorption, particularly as a result of the activity of the intracellular enzyme CYP3A4. Absorption may be transcellular (membrane diffusion, carrier-mediated, endocytosis) or paracellular, while p-glycoprotein activity in the apical cell membrane may limit bioavailability by expelling drugs from the mucosal cells. Knowledge of the absorption mechanism is important in determining formulation strategies. The different in vitro techniques used to study absorption have advantages and disadvantages. Ussing chambers can be useful to measure bidirectional transport, but most studies use simple salt media, and full tissue viability is doubtful. Caco-2 cell monolayers are human cells, but the system is static, and gives very low rates of transport, and exagerated enhancement of the paracellular route compared with small intestine. The rat everted gut sac incubated in tissue culture medium maintains tissue viability and gives reliable data, although it is a closed system. In situ perfusion gives no information on events at the cellular level, and absorption may be reduced by anaesthesia and surgical manipulation. In vivo perfusion in man, with multichannel tubes, gives valuable data, but is not practical for screening. Pharmacokinetic modelling can also give useful data such as the existence of different absorption sites. Permeability values from the literature show that for small hydrophilic molecules, which pass by the paracellular route, the improved everted sac gives values close to those for humans, while values with Caco-2 cells are orders of magnitude lower.

Methadone is used as a treatment for opiate detoxification in methadone maintenance programs. Intra- and inter-patient variations in methadone bioavailability have been observed after oral methadone treatment and this makes it difficult to predict a dosing regimen. Intestinal absorption and metabolism could explain these variations. The in vitro gut sac model was used to study the intestinal absorption of methadone, and it confirmed that methadone is a substrate for P-glycoprotein. The transport of methadone was increased in presence of P-gp inhibitors verapamil and quinidine. The appearance of a major metabolite of methadone, 2-ethylidene-1, 5-dimethyl-3, 3-diphenyl pyrrolidine (EDDP) in the gut sac contents also demonstrated the existence of intestinal metabolism of methadone.

An improved everted gut sac system has been developed in which the sacs were carefully prepared from rat small intestine and incubated in tissue culture medium. Under these conditions, the tissue showed good morphology at the electron microscope level, and was metabolically active for up to 2 h at 37 degrees C. Mannitol, an established probe of paracellular transport, was transported from the mucosal to the serosal side of the sac tissue. Excellent kinetic data showed that transport was linear up to 75 min and over a wide range of concentrations (0.025 - (10 mM). Mannitol was not detected in the tissue and transport was enhanced by EGTA, confirming the paracellular route of passage. Sacs prepared from colon also showed mannitol transport, but at a slower rate. Comparisons with Caco-2 cell monolayers showed that the everted sacs exhibited higher levels of paracellular transport than the cultured cell line. The improved everted gut sac system is an inexpensive and relatively simple technique with considerable potential as an in vitro tool to study the mechanisms, kinetics and enhancement of drug absorption across the small intestine at different sites and in the colon.

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Chondroitin sulfate (CAS 24967-93-9, CS) is a natural polymer of a disaccharide consisting of glucuronic acid and N-acetyl glucosamine which is sulfated either in the 4 or 6 position. It is administered orally as a slow acting drug to treat osteoarthritis, though there is much debate about its effectiveness and its mode of action, given that macromolecules are not normally absorbed in the gastrointestinal (GI) tract. Initially using a spectrophotometric assay, the stability of CS was tested in the presence of both tissues and lumenal contents of stomach, small intestine, cecum and colon. There was no degradation by the contents of the stomach or small intestine or in any of the tissues. Degradation only took place in the contents of the colon and particularly the cecum. Using 14C-radiolabelled CS it was shown that the cecum contents degraded CS down to the component disaccharides. The 14C-radiolabelled CS was also used to investigate the transport of CS across the different parts of the GI tract in vitro. The CS was transported across the small intestine in low amounts in the intact form, probably by the mechanism of endocytosis. In the colon and the cecum, higher amounts of radioactivity were transported, but most of the radioactivity was in the form of the degradation products, the disaccharides. This study shows that small amounts of CS may cross the upper intestine intact, but in the distal GI tract the molecule is effectively degraded, presumably by the enzymes in the intestinal flora.