Cerebral uptake of mefloquine enantiomers with and without the P-gp inhibitor elacridar (GF1210918) in mice

Mefloquine is a chiral neurotoxic antimalarial agent showing stereoselective brain uptake in humans and rats. It is a substrate and an inhibitor of the efflux protein P-glycoprotein. We investigated the stereoselective uptake and efflux of mefloquine in mice, and the consequences of the combination with an efflux protein inhibitor, elacridar (GF120918) on its brain transport. Racemic mefloquine (25 mg kg−1) was administered intraperitoneally with or without elacridar (10 mg kg−1). Six to seven mice were killed at each of 11 time-points between 30 min and 168 h after administration. Blood and brain concentrations of mefloquine enantiomers were determined using liquid chromatography. A three-compartment model with zero-order absorption from the injection site was found to best represent the pharmacokinetics of both enantiomers in blood and brain. (−)Mefloquine had a lower blood and brain apparent volume of distribution and a lower efflux clearance from the brain, resulting in a larger brain/blood ratio compared to (+)mefloquine. Elacridar did not modify blood concentrations or the elimination rate from blood for either enantiomers. However, cerebral AUCinf of both enantiomers were increased, with a stronger effect on (+)mefloquine. The efflux clearance from the brain decreased for both enantiomers, with a larger decrease for (+)mefloquine. After administration of racemic mefloquine in mice, blood and brain pharmacokinetics are stereoselective, (+)mefloquine being excreted from brain more rapidly than its antipode, showing that mefloquine is a substrate of efflux proteins and that mefloquine enantiomers undergo efflux in a stereoselective manner. Moreover, pretreatment with elacridar reduced the brain efflux clearances with a more pronounced effect on (+)mefloquine. Keywords: Mefloquine, enantiomer, stereoselectivity, P-glycoprotein, efflux protein, brain uptake, pharmacokinetics, blood–brain barrier, elacridar Introduction Mefloquine, α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol, an antimalarial agent with two asymmetric carbons, is administered orally as the racemic mixture of the two erythro-enantiomers (+)11R,2′S-mefloquine [(+)mefloquine] and (−)11S,2′R-mefloquine [(−)mefloquine] (Lariam®) in the prevention and treatment of chloroquine-resistant malaria. Serious neuropsychiatric reactions have been reported in approximately 1 : 10,000 healthy subjects receiving prophylaxis and 1 : 300 patients receiving treatment (Weinke et al., 1991; Phillips-Howard & Ter Kuile, 1995). Both enantiomers of mefloquine show similar antimalarial activity against Plasmodium falciparum, the parasite responsible for severe malaria in humans (Basco et al., 1992). The specific neurotoxic effects of separated enantiomers is not known. In case the neurotoxicity is different, it is important to study the cerebral transport of mefloquine enantiomers to determine which enantiomer is less transported into the brain. Cerebral transport of mefloquine is stereoselective. In two human cases of cerebral malaria treated with oral racemic mefloquine, plasma and brain concentrations of the (−)enantiomer were higher than those of the antipode (Pham et al., 1999). In the rat, after repeated oral administration of the racemic mixture, brain concentrations of (−)mefloquine were always higher than those of the antipode while the contrary was observed in plasma (Baudry et al., 1997). This stereoselectivity observed in the brain uptake indicates that the cerebral transport may be active. An active efflux could be responsible for this active stereoselective transport of mefloquine involving efflux proteins. The blood–brain barrier (BBB) is composed of capillary endothelial cells with efflux proteins, expressed on the luminal side of the plasma membrane. Among them, the most studied, P-glycoprotein (P-gp) and others, such as MRP and the more recently discovered Breast Cancer Resistance Protein BCRP/MXR/ABCG2 (BCRP) (Litman et al., 2001; Cooray et al., 2002; Sun et al., 2003). All of those efflux proteins are responsible for the extrusion of their substrates from the endothelial cells of the BBB back to the cerebral blood circulation. Interactions between P-gp and mefloquine have been described. It has been shown that mefloquine was able to inhibit P-gp in different drug-resistant cell lines (Lan et al., 1996; Riffkin et al., 1996; Shao et al., 1997). This inhibitory effect on P-gp was stereoselective when tested on the accumulation of vinblastine in an immortalized rat brain capillary endothelial cell line, GPNT (Pham et al., 2000) and on the retention of cyclosporin A in an immobilized P-gp liquid chromatographic stationary phase (Lu et al., 2001), (+)mefloquine being a more potent inhibitor than its antipode. In GPNT cells, in vitro efflux of racemic mefloquine was decreased by P-gp inhibitors such as verapamil and cyclosporin, indicating that mefloquine could also be a substrate of P-gp (Pham et al., 2000). No in vivo study has yet demonstrated that mefloquine is a substrate of efflux proteins in the brain. Many compounds have been shown to reverse this multidrug resistance phenomenon (Sun et al., 2003). Many commercialized drugs such as verapamil and cyclosporin A were discovered to be chemosensitizers capable of modulating the effects of these pumps. More specific inhibitors such as PSC 833 (valspodar), VX-170 (biricodar), LY335979 (zosuquidar) and GF120918 (elacridar) were later developed. Elacridar, (N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolynyl)-ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine) (also known as GF120918 or GG918) was initially described as a specific P-gp inhibitor. But when BCRP was discovered later on, many articles reported an inhibitory effect of this modulator also on BCRP, proving that elacridar was no longer specific to P-gp. Elacridar is now described as a P-gp and BCRP inhibitor (Maliepaard et al., 2001) without any effect on MRP (Evers et al., 2000). It does not modulate cytochrome metabolism (Cummins et al., 2002). Knowing that (i) mefloquine is a P-gp substrate; (ii) its cerebral transport is stereoselective; (iii) its neurotoxicity could be concentration-dependent, the objective of this study was to investigate and model the cerebral transport of mefloquine enantiomers in mice after administration of the racemic mixture used in human therapeutics and to determine whether there is a difference in brain transport between the two enantiomers. We have also investigated the consequences of the combination with the efflux protein inhibitor, elacridar on the brain transport of both enantiomers. As a modification of plasma protein binding could influence the brain uptake of mefloquine, the effect of elacridar on mefloquine protein binding was also studied.