(T1230-03-14) Regional Bile Salt and Lipid Concentrations in the Rat Small Intestine: A Comparison between the Lumen and the Mucus Layer

Purpose: The characteristics of the gastro-intestinal (GI) fluids, e.g. the pH value and the concentration of bile salts (BSs), polar lipids (PLs) and neutral lipids (NLs), directly affect the solubility and thereby the oral absorption of poorly water-soluble drugs (PWSDs). Upon in vitro evaluation of oral drug delivery systems, an improved prediction of the in vivo absorption has been found when the utilized in vitro setup mimicked the GI conditions of the animals used for the in vivo studies rather than human GI conditions [1]. Therefore, the physiological conditions in the GI tract of rats, commonly used for in vivo studies, need to be further characterized to improve the design of species-specific in vitro models intended for oral drug absorption evaluation. Additionally, while the mucus layer covering the small intestine (SI) is commonly regarded as a barrier of drug absorption [2], pilot studies indicate that mucus contains BSs, PLs and NLs at levels that could be relevant for the solubilization and absorption of orally administrated PWSDs. Thus, this study aimed to determine the pH values and concentrations of BSs, PLs, and NLs in the luminal fluids and mucus layer in the SI of rats.

Methods: Eight fasted rats were anesthetized, and the luminal pH was measured through small incisions in the SI followed by sampling of the luminal fluids (Figure 1). Another eight fasted rats were killed, the mucus layer of the SI was sampled by gentle scraping with a laboratory spoon, and the pH was measured ex vivo. The concentrations of individual BSs, PLs, and NLs in the lumen and mucus layer were determined using High-Performance Liquid Chromatography coupled with Charged Aerosol Detection.

Results: Values of pH: The luminal fluids from the duodenum to the proximal jejunum exhibited a small but significant drop in pH value (Table 1). While maintaining pH around pH 6.6 from the proximal to mid jejunum, there was a significant increase in the pH in the ileum to 7.87±0.09 (p < 0.05). In the mucus layer of the SI, pH did not change significantly from the duodenum to the mid-jejunum (Table 1), with an average pH value of around pH 6.5. As with the lumen, the pH in the mucus layer of the ileum was significantly higher when compared to the rest of the SI mucus layer (p < 0.05). Comparing the luminal values to those of the mucus layer, the pH of the luminal fluids in the SI tended towards higher values, although it was only significantly higher in the duodenum and ileum (p < 0.05). Levels of BSs, PLs, and NLs: The lumen contained higher BS concentrations when compared to the mucus layer in most of the SI, but this was only significantly in the proximal and mid jejunum (p < 0.05) (Figure 2A). While the luminal fluids had a significantly lower PL concentration in the duodenum when compared to the mucus layer, the reverse was true in the proximal jejunum (p < 0.05) (Figure 2B). There was, however, no significant difference between the two groups in the rest of the SI. The luminal NL concentration was lower than that of the mucus layer throughout the whole SI, though only significantly so in the duodenum, proximal-, and mid jejunum (p < 0.05) (Figure 2C). Generally, the studied characteristics of the rat GI tract differ from those reported in the human GI tract, both in terms of pH and levels of BSs, PLs, and NLs [3]. Higher concentrations of BSs, PLs and NLs were detected in both the lumen and mucus layer of the rat SI, as compared to the human. This could most likely affect the preclinical evaluation of oral drug absorption of PWSDs.

Conclusion: In this study, some of the key characteristics of the rat SI relevant for oral drug delivery of PWSDs were determined. The results highlight the presence of BSs, PLs, and NLs in the mucus layer of the SI at different concentrations to those of the lumen. Based on this study, it is possible to improve the design of species-specific in vitro models used in the development of new oral drug delivery systems.

References: 1. Berthelsen, R.; Klitgaard, M.; Rades, T.; Müllertz, A. In vitro digestion models to evaluate lipid based drug delivery systems; present status and current trends. Advanced Drug Delivery Reviews 142:35-49 (2019)
2. Sigurdsson, H.H.; Kirch, J.; Lehr, C.M. Mucus as a barrier to lipophilic drugs. International Journal of Pharmaceutics. 453(1):56-64 (2013)
3. Bergström CAS, Holm R, Jørgensen SA, Andersson SBE, Artursson P, Beato S, et al. Early pharmaceutical profiling to predict oral drug absorption: current status and unmet needs. European Journal of Pharmaceutical Sciences. 57:173–99 (2014).

Acknowledgments: This study was funded by Lundbeckfonden (R286-2018-2015) as well as by the Danish National Research Foundation (DNRF122) and Villum Fonden (Grant No. 9301), Center for intelligent drug delivery and sensing using microcontainers and nanomechanics (IDUN).

Figure 1. Experimental setup of the animal study. Eight fasted rats were anesthetized, and small incisions in the small intestine were used to measure the pH of the lumen in situ and sample luminal content. Another eight rats were killed, and the small intestine was divided into sections of 15 cm. Mucus was removed from each section and the pH was measured ex vivo.



Table 1. pH measurements in the lumen and the mucus layer of the rat small intestine (mean±SEM, n=8). Superscripts a and b indicate a significant difference between the groups (p < 0.05).



Figure 2. Total concentrations of bile salts (A), polar lipids (lysophospholipids, phospholipids, and sphingomyelin) (B), and neutral lipids (linoleic acid, palmitic acid, oleic acid, stearic acid, and cholesterol) (C) in the lumen (green) and mucus layer (blue) of the rat small intestine (mean±SEM, n=8). An asterisk indicates a significant difference between the two groups (p < 0.05).