In situ perfusion of intestinal segments of rodents (rats or rabbits) is frequently used to study the permeability and absorption kinetics of drugs. The biggest advantage of the in situ system compared to the in vitro techniques discussed earlier is the presence of an intact blood and nerve supply in the experimental animals. Various modifications of the perfusion technique have been studied by different investigators: single pass perfusion (Komiya et al., 1980), recirculating perfusion (Van Rees et al., 1974), oscillating perfusion (Schurgers and DeBlaey, 1984), and the closed loop method (Doluisio et al., 1969). Lennernas and colleagues (Lennernas et al., 1997; Lennernas, 1998) have extended the perfusion studies to humans. This methodology is found to be highly accurate for predicting the permeability of passively transported compounds, however the use of a scaling factor has been recommended for predicting permeability of carrier-mediated compounds.
In situ experiments for studying intestinal drug absorption involve perfusion of drug solution prepared in physiological buffer through isolated cannulated intestinal segments. Absorption is assessed based on the disappearance of drug from the intestinal lumen. Hydrodynamics of the flow of buffer through the intestinal segments can influence the absorption characteristics of the drugs due to the effect of unstirred water layer. In this technique the difference in the concentration of the inlet and outlet flow is used to calculate the permeability. The presence of an intact blood supply, nerve and clearance capabilities at the site of absorption lead to an excellent experimental system which mimics the in vivo condition. Also, the input of compounds can be closely controlled with respect to concentration, pH, flow rate, intestinal region, etc. In the single pass perfusion approach the net effective permeability (Peff) is calculated as:
where Ci and Co are the inlet and outlet concentrations at steady state of the compound in the perfusate, Qin is the inlet perfusion flow rate, and 2nRL is the mass transfer area available for absorption in the intestinal cylinder with length L and radius R.
Despite its advantages, the use of single pass perfusion method is severely limited because this method relies on the disappearance of compound from the lumi-nal side as an indication of absorption, but the rate of decrease of concentration in the perfusate does not always represent the rate of absorption of the drug into the systemic circulation (especially for compounds undergoing presystemic or luminal metabolism). The method is also limited because of its cost factor as it requires a large number of animals to get statistically significant absorption data. Relatively high amounts of test compounds are also required to perform studies (>10 mg), which is not feasible in early drug discovery. It was also demonstrated that the surgical manipulation of intestine combined with anesthesia caused a significant change in the blood flow to intestine and had a remarkable effect on absorption rate (Uhing and Kimura, 1995).
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