Studies on optimising the everted gut sac model (EGSM) in rat and possum tissue to compare selective drug movement across the intestinal wall

Abstract

The in-vitro everted gut sac model has been used extensively in the laboratory environment to investigate the transfer of orally administered drugs across the intestinal wall of rodents. This model involves excision of fresh tissue from the small intestine, which is then everted and sectioned into individual sacs and incubated under mammalian physiological conditions in medium containing the target drug. One of the limitations of this model is the need to access tissue immediately following euthanasia, greatly restricting applicability for many species. This study investigated the movement of a number of drugs across the intestinal wall in rat tissue that had been stored at 4 ºC and then utilised for studies within 60 minutes and after being stored at 24 h; as well as possum tissue that had been stored for approximately 24 h. Transfer of the target drugs fluconazole, digoxin and chloramphenicol were significantly greater in the proximal quarter of the small intestine than the distal three, and so this quarter was used for remaining experiments. A number of segments could be gained from the length of intestine from this quarter and there was no significant difference shown in transfer between any segments. The transfer rate of drugs appeared to decrease following the initial 20 minutes of incubation, and so this initial 20 minutes was investigated in the remaining experiments with rat tissue. Digoxin, a recognised P-glycoprotein substrate, demonstrated the anticipated lower transfer across the intestinal wall of the rat compared to fluconazole and chloramphenicol. Whilst the addition of 5 μg/mL verapamil increased the initial transfer of digoxin across the gut wall, the addition of 25 μg/mL verapamil resulted in significantly lower absorption of all three target drugs following 20 minutes incubation. The transfer of glucose following tissue storage for 0.5 hours was slightly significantly lower than the transfer of glucose observed in tissue stored for 24 h. The transfer of glucose across the gut wall using possum tissue was investigated at 30, 60 and 90 minutes incubation, however access to fresh tissue was problematic, and the possum EGSM did not demonstrate the anticipated transfer curve. The use of lactate dehydrogenase was examined for use as an indicator of cell toxicity, but found to be unreliable. This study concluded that whilst this model may not have future use in wildlife studies that are performed ex-situ, sufficient correlations were seen in these experiments to suggest further studies to validate the EGSM using stored tissue would be warranted.