Grapefruit juice enhances intestinal absorption of the P-glycoprotein substrate talinolol

Abstract

Grapefruit juice (GFJ) is known to affect the pharmacokinetics of various drugs, presumably mainly via inhibition of oxidative metabolism. In order to evaluate the effect of GFJ on P-glycoprotein-related transport processes, measurements of transport characteristics through Caco-2 monolayers and in vivo drug absorption studies were performed with the transported, yet not metabolized model compound talinolol. Apical-to-basolateral talinolol transport in the Caco-2 model at 1 mM racemate concentration was increased almost 3-fold when GFJ was present (S-talinolol P_eff: 0.16×10⁻⁶ vs. 0.61×10⁻⁶ cm/s without vs. with GFJ; R-talinolol P_eff: 0.19×10⁻⁶ vs. 0.71×10⁻⁶ cm/s without vs. with GFJ). In vivo in rats, doubled maximum plasma concentrations, enhanced AUC values (C_max of S-talinolol: control, 77.5 ng/ml vs. GFJ, 163.6 ng/ml; C_max of R-talinolol: control, 79.5 ng/ml vs. GFJ, 163.0 ng/ml; AUC of S-talinolol: control, 19.3 μg ml⁻¹ min vs. GFJ, 29.9 μg ml⁻¹ min; AUC of R-talinolol: control, 22.2 μg ml⁻¹ min vs. GFJ, 30.1 μg ml⁻¹ min), and decreased apparent oral clearances were found for both talinolol enantiomers when GFJ was administered together with a racemic 10 mg/kg b.w. p.o. dose. Furthermore, GFJ tended to accelerate the rate of talinolol input, but did not significantly affect terminal talinolol half-lives. It is concluded that inhibition of intestinal secretion may contribute to bioavailability enhancement upon GFJ intake.

Introduction

In the recent years various articles have been published, in which a modulating effect of nutritional constituents on drug kinetics was described. Flavonoids, particularly flavonols, were found to affect the transport of drugs and other exogenous compounds via interaction with the multidrug-transporter P-glycoprotein (P-gp) and related transporters, which are present in various tissues. The outside-directed ABC transporter P-gp as well as related transporters are involved in the reduction of cellular cytostatic drug accumulation in multidrug resistance (MDR) (Gottesman and Pastan, 1993).

Since P-gp is also present in the absorptive enterocytes of the gut, it may reduce the fraction of drug absorbed and limit bioavailability in a dose-dependent, saturable, and inhibitable manner (e.g. Wetterich et al., 1996, Langguth et al., 1997, Anderle et al., 1998). Generally, a structurally diverse group of compounds can reverse P-gp mediated MDR, and molecules with diverse structures may be substrates for P-gp mediated transport (Seelig, 1998) or may bind as ligands at different binding sites. This diversity with respect to interactions with P-gp is also observed within the group of flavonoids (Conseil et al., 1998).

With respect to potential flavonoid effects, their cellular mechanism of action has best been elucidated in cancer cells. During the past decade, various articles have been published indicating that flavonoids may not only be cytotoxic themselves (Mitrocotsa et al., 1999, Choi et al., 1999), but also an interaction may be expected between flavonoids and P-glycoprotein transporters (e.g. Phang et al., 1993, Scambia et al., 1994, Versantvoort et al., 1996, Shapiro and Ling, 1997, Maitrajean et al., 2000). Critchfield et al. (1994) identified flavonoids with no effect or increased ¹⁴C-adriamycin (¹⁴C-ADR) accumulation in P-glycoprotein expressing HCT-15 colon cells, while a number of flavonoids were reducing ADR accumulation and hence apparently upregulating and stimulating P-gp mediated efflux (most pronounced stimulation: galangin, kaempferol and quercetin), which was blocked by MDR reversing agents (such as verapamil, vinblastine and quinidine). The authors proposed allosteric effects, presumably at the 1,4-dihydropyridine-selective drug acceptor site that was described by Ferry et al. (1992) to be allosterically coupled to the vinca-alkaloid selective site, as one feasible mechanistic explanation for the stimulation. Increased, flavonol-stimulated efflux was also detected for 7,12-dimethylbenz(a)anthracene in multidrug-resistant breast cancer cells (Phang et al., 1993), while Lo and Huang (1999) examined the influence of diet on etoposide absorption in rats and found enhanced etoposide absorption in the everted sac model upon different diet/flavonoid pretreatments (flavonoid-containing natural rodent diet, quercetin).

Recently, a decreased vinblastine efflux was detected upon testing the effect of flavonoid-type grapefruit juice components in Caco-2 cells (Takanaga et al., 1998). To a number of grapefruit juice flavonoids, drug bioavailability-enhancing ability has been attributed (e.g. Bailey et al., 1991, Garg et al., 1998, Fuhr, 1998). Yet, the underlying mechanism appeared to be largely related to inhibition of biotransformation (inhibition of the cytochrome P-450 isoenzyme CYP3A4) as opposed to inhibition of drug counter-transport. Their influence on P-glycoprotein is still unclear, particularly regarding the in vivo relevance of this potential interaction mechanism. The inhibition of intestinal, but not hepatic CYP3A enzymes has been hypothesized as major mechanism for the enhancement of the fraction that reaches the systemic circulation after p.o. intake of CYP3A4 substrates together with grapefruit juice (Schmiedlin-Ren et al., 1997). 6′,7′-Dihydroxybergamottin, a constituent of grapefruit as well as the Seville orange juice — yet not of common orange juice — was found to inhibit CYP3A4 in enterocytes, but not to inhibit P-glycoprotein (Edwards et al., 1999). No general influence of orange juice — as opposed to grapefruit juice — on the pharmacokinetics of drugs has been found so far.

On the basis of the results published with respect to the influence of grapefruit juice and its constituents on drug transport by various authors, Soldner et al. (1999) tested a number of CYP3A/P-gp substrates using MDCK-MDR1 cells, hypothesizing that bioavailability-enhancing effects should be higher for combined substrates than for compounds that are solely CYP3A substrates. In their in vitro model, an increased efflux ratio was detected for all compounds (e.g. cyclosporin, felodipine, nifedipine) when grapefruit juice was added. As conclusion these authors attributed increased intestinal cytoprotectivity upon grapefruit juice digestion, since grapefruit juice was found to enhance P-gp mediated transport, supporting the data of Phang et al. (1993) who reported the unique property of stimulating drug transport by P-glycoprotein in vivo for three selected flavonoids.

Because of the overlapping CYP3A4/P-gp substrate specificities observed for a multitude of drugs (Wacher et al., 1995) it may be difficult to differentiate between metabolism and transport inhibition or to detect transport activation. Hence, we selected a compound that is characterized by a negligible metabolic clearance as model compound for P-gp mediated transport processes, the racemically administered β-adrenoceptor antagonist talinolol (Spahn-Langguth et al., 1998).

The aim of the present study was to elucidate the overall relevance of interactions of grapefruit juice components as present in a marketed mixture on intestinal P-gp mediated transport in Caco-2 cell monolayers and on in vivo bioavailability of the non-cytochrome P-450 substrate, yet P-gp substrate talinolol.