The application of P-gp inhibiting phospholipids as novel oral bioavailability enhancers — An in vitro and in vivo comparison

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Abstract

The efflux transporter P-glycoprotein (P-gp) significantly modulates drug transport across the intestinal mucosa, strongly reducing the systemic absorption of various active pharmaceutical ingredients. P-gp inhibitors could serve as helpful tools to enhance the oral bioavailability of those substances. As a membrane-associated protein P-gp is surrounded and influenced by phospholipids. Some synthetic phospholipids have been found to strongly reduce P-gp's activity. In this study two representative phospholipids, 1,2-dioctanoyl-sn-glycero-3-phosphocholine (8:0 PC) and 1,2-didecanoyl-sn-glycero-3-phosphocholine (10:0 PC), were compared with Tween® 80 and Cremophor® EL, both commonly used surfactants with P-gp inhibitory properties. Their influence on the cellular transport of the P-gp substrate rhodamine 123 (RH123) was examined using Caco-2 cell layers. In addition, fluorescence anisotropy measurements were performed in order to investigate their effect on membrane fluidity. Finally, we compared the phospholipids with Tween® 80 and the competitive P-gp inhibitor verapamil in an in vivo study, testing their effects on the oral bioavailability of the P-gp substrate drug ritonavir. Both phospholipids not only led to the strongest absorption of RH123, but a permeability enhancing effect was detected in addition to the P-gp inhibition. Their effects on membrane fluidity were not consistent with their P-gp inhibiting effects, and therefore suggested a more complex mode of action. Both phospholipids significantly increased the area under the ritonavir plasma level curve (AUC) within 150 min by more than tenfold, but were inferior to Tween® 80, which showed superior solubilizing effects. Finally, these phospholipids represent a novel substance class showing a high permeabilization potential for P-gp substrates. Because of their physiological structure and intestinal degradability, good tolerability without systemic absorption is expected. Formulating P-gp substrates with an originally low oral bioavailability is a difficult task, requiring concerted interplay of all excipients. P-gp inhibiting phospholipids offer a new tool to help cope with these challenges.

Introduction

The membrane associated efflux transporter P-glycoprotein (P-gp), also named multidrug resistance protein one (MDR1) or ABCB1, is the best characterized and most clinically relevant representative of the ATP-binding cassette (ABC) superfamily. P-gp primarily affects xenobiotic compounds, with a broad range of substrates. It consists of two transmembrane domains (TMDs) and two cytosolic nucleotide binding domains (NBDs). Although the transport mechanism has not been completely elucidated to date, one of the most commonly proposed explanations is that, after partitioning into the cell membrane, a P-gp substrate reaches the transporter's binding pocket from the inner membrane leaflet and triggers a large conformational change of the TMDs. This in turn presents the substrate to the outside surface, where the substrate binding affinity to P-gp decreases, and causes an efflux into the extracellular lumen or into the outer membrane leaflet. This process is dependent on ATP hydrolysis by the NBDs providing the necessary energy (Aller et al., 2009, Zolnerciks et al., 2011).

Most current research has focused on fighting cancer types that overexpress MDR1, which leads to their resistance to anti-cancer agents and in turn limits treatment success. However, P-gp is also physiologically expressed in the apical membrane of enterocytes in the small intestine. It significantly modulates drug transport across the intestinal mucosa, strongly reducing the systemic absorption of various affected substances (Giacomini et al., 2010). Plenty of active pharmaceutical ingredients (APIs) in different pharmaceutical substance classes display P-gp substrate properties resulting in a potential reduction in oral bioavailability (Marzolini et al., 2004). The consequence of this is higher API doses and intestinal side effects due to a large amount of non-absorbed substance, as well as increased therapy costs. One of these APIs is the HIV protease inhibitor ritonavir. Even when used in low dose regimes, gastrointestinal side effects remain present and impair patient compliance (Cooper et al., 2003). Intestinal P-gp inhibitors provide a promising field of application to reduce such problems.

With increasing selectivity and potency P-gp inhibitors can be classified into three generations. First generation substances like verapamil or cyclosporine A were originally developed for other indications, while second generation substances are represented by more selective or less toxic derivatives from the first generation, e.g. PSC 833 (a P-gp inhibiting cyclosporine derivative without immunosuppressive properties), and third generation substances like zosuquidar were designed as highly potent P-gp inhibitors with no other intended target (Constantinides and Wasan, 2007). Surfactants as a further substance class are highly promising especially for the enhancement of the oral bioavailability of drugs. Several of these are approved excipients and commonly used as emulsifiers, solubilizers or wetting agents. Their ability to increase membrane fluidity and reduce lateral packing density of membrane lipids is assumed to force a conformational change in P-gp and cause its inhibition (Li-Blatter et al., 2009, Rege et al., 2002). However, this proposed mechanism remains controversial and a definite explanation which covers all circumstances is still missing (Sharom, 2014). Nevertheless, due to the broad range of substances in this class and additional bioavailability enhancing properties, e.g. solubilization, as well as their good tolerability and absence of further pharmaceutical activities, this approach has been a popular subject of study for decades (Constantinides and Wasan, 2007, Cornaire et al., 2004, Drori et al., 1995, Dudeja et al., 1995, Martin-Facklam et al., 2002, Seelig and Gerebtzoff, 2006, Woodcock et al., 1990, Woodcock et al., 1992, Zhang et al., 2003).

Phospholipids (PLs) are underrepresented in this field of research. As a major component of biomembranes they influence associated proteins and transport processes. The interplay of natural PLs with P-gp is complex and has been extensively reviewed elsewhere (Sharom, 2014). Furthermore, PLs have diverse uses as excipients for a variety of application routes and formulation types such as in liposomes, mixed micelles, (nano)emulsions, self-emulsifying drug delivery systems (SEDDS), solid lipid nanoparticles, suspensions and PL-drug complexes (Fricker et al., 2010). In previous studies by our group the effect of several synthetic PLs on P-gp was investigated using transport studies, calcein accumulation and P-gp ATP-ase activity tests (Simon and Schubert, 2012). Promising candidates which significantly reduced P-gp activity were identified. Docking studies also showed strong binding affinity of some PL representatives in the substrate binding pocket, suggesting a direct mode of interaction (Lucas et al., 2013, Simon, 2013).

To continue these studies, we set our focus on the two most effective candidates, 1,2-dioctanoyl-sn-glycero-3-phosphocholine (8:0 PC) and 1,2-didecanoyl-sn-glycero-3-phosphocholine (10:0 PC) (structures in relation to natural PLs are given in Fig. 1). The aim of this study was to compare these PLs with Tween® 80 and Cremophor® EL, which are both approved P-gp inhibition related non-ionic surfactants (Rege et al., 2002). The influence of the PLs on the transport of rhodamine 123 (RH123), a model P-gp substrate (Forster et al., 2012), through Caco-2 cell layers was tested and their impacts on membrane fluidity were investigated by fluorescence anisotropy measurements. In addition, we performed oral bioavailability studies in male Wistar rats to elucidate the effects of the PLs on ritonavir uptake. Tween® 80 and the competitive 1st generation P-gp inhibitor verapamil were also included in the study for comparison. The interplay between P-gp inhibition and ritonavir solubilization was addressed by dissolution testing.

Section snippets

Cell cultivation

Caco-2 cells (DSMZ, Braunschweig, Germany), passage 17–22, were grown in culture dishes (Falcon BD Biosciences®, Heidelberg, Germany) at 37 °C and 90% rh under 5% CO2 atmosphere in cell culture medium (CCM) consisting of Dulbecco's modified Eagle's medium (DMEM) with 3.7 g/l NaHCO3, 4.5 g/l glucose and stable glutamine, 10% heat inactivated fetal calf serum, 1% l-alanyl-l-glutamine 200 mmol/l, 1% non-essential amino acids (100 × concentrate) and 1% sodium pyruvate 100 mmol/l. For transport studies,

Lipid formulation characterization

All 10:0 PC formulations yielded a liposomal dispersion with a mean hydrodynamic particle diameter between 60 and 170 nm and a PDI between 0.09 and 0.23. EPC/Chol (blank and DPH labeled) liposomes possessed a diameter of 179 ± 26 nm with a PDI of 0.195 ± 0.093. Particles of 8:0 PC were sized by Nanoparticle Tracking Analysis with a mean diameter of 154 ± 97 nm (Fig. 2). Cryo TEM analysis (Fig. 3) showed different types of 8:0 PC associates from small micelles and vesicles to larger coherent structures.

Conclusion

The HIV protease inhibitor and booster agent ritonavir is a commonly used P-gp substrate in many anti-retroviral therapy regimes. A serious problem with these therapies are gastro-intestinal side effects, impairing patient compliance and therapy adherence in a dose-dependent manner (Cooper et al., 2003). P-gp inhibitors can be used to reduce the doses needed to maintain the therapeutic effect with less gastrointestinal side effects caused by non-absorbed drug. In our study we have shown that

Acknowledgements

The authors want to thank Phospholipid e.V. (Heidelberg, Germany) for financial support. Cryo TEM pictures were taken by Sabine Barnert (Department of Pharmaceutical Technology & Biopharmacy, University of Freiburg). Assistance in formulation preparation and dissolution testing was provided by Christina Barth, Friederike Schmid, and Kira Schwarz. Monika Maurer assisted in UPLC/MS/MS analysis of ritonavir in plasma samples. We thank Kathrin Züfle for helpful discussions. Part of this work was

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