Intestinal Perfusion With Mesenteric Blood Sampling in Wild-Type and Knockout Mice

Abstract

ABSTRACT:In the present study, we successfully downscaled, for the first time, the in situ intestinal perfusion technique with mesenteric blood sampling from rat to mouse. To evaluate the feasibility of this approach, we assessed the apparent permeability (P app ) of mouse intestine for a set of marker compounds [atenolol, paracellular transport; metoprolol, transcellular transport; talinolol, P-glycoprotein (P-gp)-mediated efflux] in both wild-type and P-gp-deficient mice. In wild-type mice, the observed P app values for atenolol (1.8 ؎ 0.3 ؋ 10 ؊6 cm/s) and metoprolol (50.2 ؎ 20.1 ؋ 10 ؊6 cm/s) were not significantly affected by inclusion of the P-gp inhibitor verapamil. In contrast, the P app value for talinolol (0.9 ؎ 0.3 ؋ 10 ؊6 cm/s) increased 5-fold in the presence of verapamil. The similarity between these values and previously determined P app values in rats indicates comparable passive barrier functions and P-gpmediated efflux transport between mice and rats. In comparison with wild-type mice, the apparent permeability in P-gp-deficient mdr1a/1b(؊/؊) mice was significantly altered for talinolol (7-fold increase) but not for atenolol or metoprolol. Because of the availability of knockout mice, the intestinal perfusion technique with mesenteric blood sampling in mice may become an important tool to elucidate the role of intestinal metabolism and active transport in drug absorption during preclinical drug evaluation.Accurate assessment of intestinal permeability for drugs and drug candidates intended for oral administration is crucial to making rational decisions according to the absorption potential and to unraveling absorption mechanisms that may explain limited and/or variable bioavailability. For instance, intestinal metabolism and active transport (uptake or efflux) might affect the rate of transepithelial drug transport and mediate drug-drug and drug-food interactions, possibly causing variable drug disposition, decreased efficiency, or unwanted side effects (Fleisher et al., 1999;Custodio et al., 2008). Among the variety of permeability models available to study transepithelial transport, the in situ intestinal perfusion technique with mesenteric blood sampling in animal models (mostly rats) is the appropriate approach when biorelevance and accurate prediction rather than high-throughput screening are required. The technique provides the best simulation of the in vivo situation (including relevant barrier functions and sink conditions. In particular, the biorelevant expression of intestinal enzymes and transporters makes the model well suited to elucidate their impact on the absorption process, even though interspecies differences may complicate extrapolation to humans.

In Situ Intestinal Perfusion in Knockout Mice Demonstrates Inhibition of Intestinal P-Glycoprotein by Ritonavir Causing Increased Darunavir Absorption

“…In the present study, 10 times higher concentrations were used to explore the transport characteristics of DRV using Caco-2 cells. In addition, by using the recently developed in situ intestinal perfusion technique (Mols et al, 2009), we investigated the influence of P-gp on the uptake of DRV from the intestinal lumen at concentrations that are more relevant for the in vivo situation, based on the solubility of DRV in intestinal fluids.…”

“…Experiments were performed using male FVB and mdr1a/1b(Ϫ/Ϫ) FVB mice (the Netherlands Cancer Institute, Amsterdam, The Netherlands). The setup for the in situ perfusion experiments in mice has previously been described by Mols et al (2009). The perfusion experiments were performed using an open-loop set-up of the perfusate.…”

“…In addition, we explored the effect of fasted state simulated intestinal fluid (FaSSIF) and RTV coadministration (75 M) on darunavir uptake using Caco-2 monolayers. We investigated intestinal absorption using the recently developed in situ intestinal perfusion technique in wild-type and mdr1a/1b(Ϫ/Ϫ) mice by measuring the appearance of darunavir in the mesenteric blood (Mols et al, 2009). To the best of our knowledge, this is the first time that intestinal absorption of DRV is explored in knockout mice.…”

Boosting of HIV Protease Inhibitors by Ritonavir in the Intestine: The Relative Role of Cytochrome P450 and P-Glycoprotein Inhibition Based on Caco-2 Monolayers versus In Situ Intestinal Perfusion in Mice

“…Experiments were performed using male NMRI mice (Janvier, France). The setup for the in situ perfusion experiments in mice has been described previously by Mols et al (2009). The perfusion experiments were performed using an open-loop setup.…”

“…It offers a better prediction of human absorption than cellbased assays (Salphati et al, 2001) because of the following: 1) an intact intestinal mucosa, nerve system, and blood flow; 2) the presence of sink conditions; and 3) the expression of all enzymes and transporters. The rat is the standard animal used for this technique, but Mols et al (2009) introduced the use of mice in the in situ intestinal perfusion technique with mesenteric blood sampling. This offers the possibility to use knockout and knockin animals or perform in situ absorption studies when only small quantities of test compound are available.…”