Improved understanding of the effect of food on drug absorption and bioavailability for lipophilic compounds using an intestinal pig perfusion model

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Abstract

The purpose of this study was to investigate the relative importance of mechanisms behind the effect of food on the intestinal absorption and bioavailability for low solubility compounds by applying a porcine single-pass perfusion model. Nanoparticle suspensions of the model compounds, danazol and cyclosporine were perfused through the jejunum in isotonic fluid alone (control) and isotonic fluid with a P-glycoprotein (P-gp) inhibitor (verapamil) or dietary and endogenous lipids added. The drugs were also administered as saturated solutions in the isotonic fluid containing lipids. Administration of cyclosporine together with verapamil increased the absorption compared to the control (1.6 times) suggesting an effect on jejunal permeability. However, addition of dietary lipids to the media led to a 50% reduction in the absorption of cyclosporine indicating lack of major effects by P-gp inhibition by lipids in vivo. The absorption of danazol was increased (2.6 times) when administered as a nanosuspension in lipid containing media compared to the control, but decreased (60%) when administered as a solution in the same media. This shows how important dissolution of the drug nanoparticles is in drug absorption. The difference in the effect of lipids in the absorption of cyclosporine and danazol when administered as nanosuspensions may be due to different distribution to the colloidal structures present in the media, thereby rendering the drugs’ different diffusion rates in the perfused segment. In conclusion, solubilisation seems to be a more important factor than P-gp inhibition as an explanation for the food–drug interaction observed for several low solubility drugs. In addition, the partition into different colloidal structures seems to play a major role in the dissolution and absorption of poorly soluble drugs.

Introduction

According to a WHO list (Kasim et al., 2004, Lindenberg et al., 2004), the most highly hydrophobic compounds belong to Class II (low solubility/high permeability) of the Biopharmaceutical Classification System (BCS), and few to Class IV (low solubility/low permeability). Successful approaches that have been adopted to increase the absorption of Class II drugs include the use of lipid-based formulations and concomitant intake with food (Caliph et al., 2000, Charman et al., 1993a). Proposed mechanisms for the increased absorption using these approaches are increased drug solubility and dissolution, lymphatic uptake (Ueda et al., 1983, Porter et al., 1996), decreased first pass metabolism (Ingwersen et al., 1993) and inhibition of efflux transporters in the apical membrane (Wu and Benet, 2005). Recent investigations in simulated and human intestinal fluids show that the solubility of poorly soluble drugs increased by solubilisation in the micelles present in the fed state small intestinal fluid (Persson et al., 2005, Pedersen et al., 2000). However, the importance of solubilisation in micelles in the intestinal lumen for bioavailability have been challenged. Recent in vitro studies in Caco-2 cells show that both taurocholate and free fatty acids increase the absorptive permeability of cyclosporine, as suggested by inhibition of apical P-glycoprotein (P-gp) efflux in the enterocyte (Ingels et al., 2002, Deferme et al., 2003). As yet, no mechanistic in vivo studies to elucidate the relative role of P-gp and/or solubilisation for increased absorption and drug bioavailability when administered with food have been carried out. A clear understanding of the mechanism by which simultaneous food intake and lipid formulations influence the absorption of Class II drugs would increase the possibility for more rational formulation approaches and improved predictability of in silico/in vitro assessments.

To determine the relative importance of P-gp inhibition and solubilisation on the absorption of Class II substances, the solubility of two model drugs, cyclosporine and danazol, were investigated in an intestinal single-pass perfusion pig model after administration of different test solutions (Petri et al., 2006). Both cyclosporine and danazol were shown to have high intestinal permeability values and low aqueous solubility, and therefore belong to the BCS class II (Table 1). Earlier in vitro results showed that cyclosporine is a substrate of both P-gp and CYP3A4 (Gan et al., 1996), while danazol is only a substrate for CYP3A4. The absorption, metabolism information and physical–chemical parameters of the model substances are displayed in Table 1. In the present study, nanoparticle suspensions of cyclosporine and danazol were single-pass perfused through a jejunal segment in isotonic fluid alone or with the addition of a P-gp inhibitor or dietary and endogenous lipids. In addition to the drug suspensions, saturated drug solutions in isotonic fluid containing lipids were perfused in the pig in order to determine the effect of food on the in vivo dissolution of cyclosporine and danazol. The different mechanisms for intestinal absorption process of cyclosporine and danazol from the four administrations given are illustrated in Fig. 1a–d.

The aim of the present in vivo study was to investigate the relative importance of mechanisms behind the effect of food on the intestinal absorption and bioavailability for low solubility compounds by applying a jejunal single-pass perfusion model in porcine. A special focus of the current study was to compare the importance of inhibition of apical efflux and micelle solubilisation.

Section snippets

Preparation of perfusion solutions

Four different test formulations (Table 2) of cyclosporine and danazol (Sigma–Aldrich, Germany, 98 and 95% pure, respectively) were prepared, as follows:

  • Control (C). Nanoparticle suspension of cyclosporine (1.25 mg/ml) and danazol (3 mg/ml) in isotonic fluid.

  • Treatment 1 (T1). Nanoparticle suspension of cyclosporine (1.25 mg/ml) in isotonic fluid, containing the P-gp inhibitor verapamil (0.5 mg/ml) (Knoll AG, Ludwigshafen, Germany).

  • Treatment 2 (T2). Nanoparticle suspension of cyclosporine (1.25 

Colloidal particle size determination

The particle size measurements are presented in Table 3. Only particles making up >1% of the mass were considered to be significant. In the isotonic fluid containing lipids, particles ranging from 1 to 316 nm in size were found. Within this interval, at least two different size distributions were identified, with mean values of 2.4 ± 0 and 122 ± 15 nm (n = 4), respectively. Neither the addition of the drug substances, nor a single-pass jejunal perfusion through the intestine affected the size

Discussion

In the present study in pigs cyclosporine had a high jejunal permeability, which agreed with corresponding human in vivo data from an in vivo jejunal perfusion study (Chiu et al., 2003). In addition, verapamil inhibited the P-gp-mediated apical efflux and further increased the intestinal Peff for cyclosporine in the pig jejunum. Thus, there seems to be an effect of P-gp inhibition by verapamil on the intestinal absorption rate of cyclosporine, verifying that the present pig model was sensitive

Acknowledgements

We would like to thank Martin Herrström for assistance in the perfusion study and Frida Rangbratt for performing the particle size measurements. We are also very grateful for valuable discussions with Lennart Lindfors, Erik Söderlind and Jan-Erik Löfroth in the planning of the study and discussion of the results.

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