Adaptation of the Kirkstall QV600 LLI Microfluidics System for the Study of Gastrointestinal Absorption by Mass Spectrometry Imaging and LC-MS/MS

Abstract

Absorption studies on oral drugs can be difficult due to the challenge of replicating the complex structure and environment of the GI tract. Drug absorption studies can be conducted using in vivo and ex vivo animal tissue or animal-free techniques. These studies typically involve the use of Caco-2 cells. However, Caco-2 cells do not incorporate all the cell types found in intestinal tissue and lack P450 metabolizing enzymes. The QV600 LLI system is a microfluidics system designed for use with cell culture. Here, it has been adapted to house appropriate sections of ex vivo porcine tissue to act as a system that models the duodenum section of the small intestine. A pH regulated solution of Atorvastatin was flowed over the apical layer of the GI tissue and a nutrient solution flowed over the basal layer of the tissue to maintain tissue viability. The tissue samples were snap-frozen, cryosectioned, and imaged using MALDI Mass Spectrometry Imaging (MSI). A proof-of-concept study on the effect of excipients on absorption was conducted. Different concentrations of the solubilizing agent were added to the donor circuit of the QV600 LLI. The amount of Atorvastatin in the acceptor circuit was determined to study the effect of the excipient on the amount of drug that had permeated through the tissue. Using these data, Papp, pig values were calculated and compared with the literature.

Keywords: Atorvastatin; mass spectrometry imaging; microfluidics; polysorbate 80.

Figure 1

Adaptation of the QV600 LLI (Kirkstall Ltd.) microfluidics cell culture chamber to hold ex vivo tissue.

Figure 2

Using the QV600 LLI, intact porcine small intestinal tissue was treated with 0.5 mg/mL Atorvastatin over a 6 h period to investigate drug absorption. (A) A scanned image of the intestinal tissue section taken using a Super Coolscan 5000 ED Film Scanner with the apical layer facing upwards. (B) A MALDI-MS image showing cholesterol [Chol+H-H₂O]⁺ at m/z 369 in red. (C) A MALDI-MS image showing the Peyer’s patches at m/z 389 in blue. (D) A MALDI-MS image showing cholesterol [Chol+H-H₂O]⁺ at m/z 369 in red and Peyer’s patches in blue; overlapping ions are shown in pink. (E) A MALDI-MS image showing the sodium adduct of atorvastatin at m/z 581 in green. (F) A MALDI-MS image showing the sodium adduct of atorvastatin at m/z 581 in green and cholesterol [Chol+H-H₂O]⁺ at m/z 369 in red. (G) A MALDI-MS image showing sodium adduct of atorvastatin (m/z 581) in green and Peyer’s patches (m/z 389) in blue. (H) A MALDI-MS image showing the sodium adduct of atorvastatin at m/z 581 in green, cholesterol [Chol+H-H₂O]⁺ at m/z 369 in red and Peyer’s patches in blue; overlapping ions are shown in pink.

Figure 3

Using the QV600 LLI, porcine small intestinal tissue with the muscle-serosal layer removed was treated with 0.5 mg/mL Atorvastatin over a 6 h period to investigate drug absorption. A MALDI-MS image was generated showing cholesterol [Chol+H-H₂O]⁺ at m/z 369 in red and protonated molecule of atorvastatin at m/z 559 in green.

Figure 4

The effect of super refined polysorbate 80 LQ on the absorption of Atorvastatin in the duodenum section of ex vivo porcine small intestine (n = 3).