ABSTRACT

Dietary effects on host-microbe interactions in pharmaceutical development - a practical example.

Henrik Rasmussen, Derek Grant, Tor Inge T�nnessen, Peyman Mirtaheri, Hubert Dirven, Helge Johnsen and Tore Midtvedt


Amersham Health AS, Oslo, Norway; Rikshospitalet, Oslo, Norway and Karolinska Institute, Stockholm, Sweden.

Practical effects of dietary host-microbe interactions in animal research are often elusive and supplying a 'complete diet' from a reputable supplier is often the solution to an inherently complex issue. Gas-carrier contrast agents (GCAs) is an example from pharmaceutical R&D, where diet is an essential parameter to consider in development studies. GCAs are free flowing gas microbubbles in clinical use, which acts as ultrasound contrast agents. However, upon a single iv. administration of GCAs, characteristic and severe lesions are observed in a distinct area of the cecum/colon and liver in mice and rats, but not in other species. All GCAs have the same toxic effects in mice, and gas content appears to be essential for the toxicity. By use of a modified fluorescein flowmetry (FF) method, irregular fluorescence of the cecum and liver were observed after GCA dosing in mice. Gas bubbles $50µm were observed to occlude the vasculature of the cecal wall in and around the hypofluorescent areas and were also seen moving with the blood stream in the portal vein. In germfree mice, normal fluorescein distribution and no lesions were observed by FF. When maintained on standard rodent diets, luminal and serosal PCO2 levels in the cecum were up to 55 ±4 kPa and 27 ±3kPa (mean ±SE) in conventional mice, 52 ±3 kPa and 9 ±1kPa in rats and 37 ±2 kPa and 9 ±0 kPa in guinea pigs. The luminal and serosal PCO2 levels in the colon were 81 ±19 kPa and 4 ±1 kPa in dogs with intestinal content and 14 ±3 kPa and 6 ±1 kPa in dogs with no/little intestinal content. PCO2 levels of 8-10 kPa were measured in lumen and serosa of germfree mice and mice where the cecal content had been removed by saline flushing. Serosal PCO2 of dogs and mice was significantly lower and higher, respectively, than in all other species. After death, serosal PCO2 of the cecum/colon increased rapidly in rats and mice, slowly in guinea pigs and very slowly in dogs. These data indicate that gas supersaturation occurs naturally in the cecal wall of mice and possibly also rats, due to the thin cecal wall and insufficient vascular clearance of the CO2 produced by bacterial fermentation of the dietary substrate (primarily dietary fibers). Gas nuclei, such as GCAs, will grow instantly when exposed to conditions of gas supersaturation and will consequently occlude the vessels in the cecal wall and cause gas embolisation of the liver. To confirm these findings, experiments were performed in conventional mice maintained on special diets. When a diet, composed of 73% glucose as the only carbohydrate source, was used, PCO2 in the cecal lumen and serosa was 23 ±1 and 12 ±1 kPa, respectively and no lesions occurred after GCA administration. Also, when 0.5% Streptomycin in the drinking water was added to a conventional diet (e.g. 14% dietary fibers, 46% starch), no lesions occurred after GCA dosing. These results may explain why lesions of the cecum occur shortly after administration of GCAs in mice and rats, while GCAs are well tolerated in guinea pigs, dogs and humans.
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