MDR1A (ABCB1)-deficient CF-1 mutant mice are susceptible to cerebral malaria induced by Plasmodium berghei ANKA.

Under experimental conditions, Plasmodium berghei infection causes cerebral malaria (CM) in susceptible strains of mice such as C57BL/6 and CBA/Ca, whereas BALB/c or DBA/2J strains serve as a model for CM-resistant mice. The aim of the present study was to investigate the susceptibility of the CF1 mouse strain, carrying a spontaneous mutation of the mdr1a gene, to infection with Plasmodium berghei ANKA (PbA). The mdr1a gene codes for P-glycoprotein (P-gp/ABCB1), an efflux pump that is one of the major components of the blood-brain barrier. P-gp effluxes a broad range of xenobiotics from the brain to blood, preventing accumulation and toxicity in the central nervous system. CFI mdr1a (-/-) mice are used to investigate drug transport by efflux pumps. Because many antimalarial agents are effluxed by P-gp (mefloquine, quinine), it was important to determine whether CF1 mice can develop cerebral malaria to predict drug toxicity during cerebral malaria. Our work showed that CF1 mdr1a (-/-) mice are susceptible to PbA. CF1 and C57BL/6N mice (the reference strain) infected with PbA have similar profiles with regard to clinical signs, brain histological lesions, and brain macrophagic activation observed by immunohistological methods.

Interactions between the dopamine agonist, bromocriptine and the efflux protein, P-glycoprotein at the blood-brain barrier in the mouse.

Bromocriptin (BCT) is a dopaminergic receptor agonist, poorly transported through the blood-brain barrier (BBB) and responsible for central side effects. Interactions between BCT and the efflux protein, P-glycoprotein (Pgp), have been described in vitro but nothing is known in vivo nor at the BBB level. At the BBB, in vivo, we investigated BCT as (i) a Pgp substrate by comparing the brain uptake in CF1 mdr1a(-/-) and mdr1a(+/+) mice with or without inhibitors of Pgp (valspodar, elacridar); (ii) a Pgp inducer by looking at the effect of repeated doses of BCT on cerebral uptake of digoxin and comparing it to the effect of dexamethasone and rifampicin; (iii) a Pgp inhibitor by determining the effect of a single dose of BCT on cerebral uptake of digoxin and comparing it to the effect of valspodar. CF1 mdr1a(-/-) mice showed much higher brain uptake of BCT than CF1 mdr1a(+/+) mice and brain uptake of BCT was higher in CF1 mdr1a(+/+) mice pre-treated with valspodar or elacridar indicating that BCT is a Pgp substrate at the BBB level. Brain uptake of digoxin was not modified in CF1 mdr1a(+/+) mice pre-treated with a single dose or repeated doses of BCT, indicating that BCT is neither a Pgp inductor nor a Pgp inhibitor at the BBB in the chosen experimental setting. In vivo, at the mouse BBB level and in our experimental conditions, bromocriptin is a Pgp substrate but is not a Pgp modulator.

Development of an assay method for the detection and quantification of protease and non-nucleoside reverse transcriptase inhibitors in plasma and in peripherical blood mononuclear cells by liquid chromatography coupled with ultraviolet or tandem mass spectrometry detection.

We present a simple chromatographic method to detect and quantify protease inhibitors (PI), metabolites and non-nucleoside reverse transcriptase inhibitors (NNRTIs) in human plasma of HIV-1 infected patients and in peripheral blood mononuclear cells (PBMCs) using either liquid chromatography coupled with ultraviolet (LC-UV) or liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). A solid-liquid extraction was carried out on 500 microl of plasma as pre-treatment. Calibration curve ranges were from 50 (100) to 5000 ng/ml (indinavir). PBMC pellets from 7 ml of blood were lysed with methanol/tris with a calibration curve ranging from 0.25 to 250 ng/pellet. Simple modifications in the mobile phase composition (slight increase of ammonium acetate concentration and addition of methanol for LC-UV) easily linked the two analytical systems.

Effect of efavirenz on intestinal p-glycoprotein and hepatic p450 function in rats.

PURPOSE: P-glycoprotein (P-gp) and cytochrome P450 (P450) affect drug disposition. Efavirenz (EFV) is an anti-HIV drug used in combination. Since most anti-HIV medications are substrate and modulators of P-gp and/or P450, we investigated the effects of EFV on intestinal P-gp and hepatic P450 function to predict drug interactions. METHODS: (i) The effect of EFV on rat intestinal P-gp function was studied on everted gut sacs and in situ intestinal perfusion. EFV was orally administered (150 mg/kg) for 6 days. Then, rhodamine 123 was used as a P-gp substrate and verapamil as an inhibitor. P-gp function was evaluated by the difference between rhodamine 123 transport with and without verapamil. (ii) The effect of EFV on rat hepatic P450 metabolism was investigated using hepatic microsomes, prepared from rats pretreated or not with EFV. RESULTS: In everted gut sacs, P-gp function was not modified and in the in situ intestinal perfusion, rhodamine 123 clearance was not affected by EFV. Concentrations of the metabolites, 1-OH midazolam and 4-OH midazolam were higher in EFV pretreated rats than those in the control group. CONCLUSION: EFV should not modify intestinal absorption of co-administered substrates of P-gp, but could decrease plasma concentrations of co-administered drugs metabolized by P450.

Evaluation of pharmacokinetic interactions after oral administration of mycophenolate mofetil and valaciclovir or aciclovir to healthy subjects.

OBJECTIVE: To investigate a potential pharmacokinetic interaction between mycophenolate mofetil (MMF) and aciclovir or valaciclovir. STUDY DESIGN AND PARTICIPANTS: Fifteen healthy subjects were enrolled in a prospective, randomised, open-label, single-dose, cross-over study conducted at a single centre. Subjects received each of the following five oral treatments: (i) aciclovir 800 mg alone; (ii) valaciclovir 2 g alone; (iii) MMF 1 g alone; (iv) valaciclovir 2 g + MMF 1 g; and (v) aciclovir 800 mg + MMF 1 g. The following pharmacokinetic parameters were estimated for aciclovir, mycophenolic acid (MPA) and its inactive glucuronide metabolite (MPAG) from the plasma concentration-time data using noncompartmental methods: area under the concentration-time curve from zero to infinity (AUC infinity), terminal elimination half-life (t1/2z), peak concentration (Cmax) and time to Cmax (tmax). The renal clearance (CLR) of aciclovir was also calculated. These parameters were compared when aciclovir or valaciclovir were coadministered with MMF relative to aciclovir, valaciclovir or MMF given alone. RESULTS AND DISCUSSION: Aciclovir Cmax, tmax and AUC infinity were significantly increased by 40%, 0.38 hour and 31%, respectively, following coadministration of aciclovir and MMF, whereas aciclovir t1/2z was significantly decreased by 11%. Following coadministration of valaciclovir and MMF, aciclovir pharmacokinetic parameters were not significantly modified except for tmax (about 0.5 hour shorter with MMF). MPA and MPAG pharmacokinetic parameters were not significantly modified following coadministration of MMF with valaciclovir or aciclovir except for MPAG AUC infinity, which was decreased by 12% with valaciclovir. Our results are similar to those reported in the literature, except for MPAG AUC. In urine, following coadministration of aciclovir and MMF, aciclovir CLR was significantly decreased by 19%. Competition between MPAG and aciclovir for renal tubular secretion could partly explain this phenomenon. Following coadministration of valaciclovir and MMF, aciclovir CLR was not significantly modified. CONCLUSION: In healthy subjects, interactions are observed after coadministration of MMF and aciclovir, but the extent of the interactions is unlikely to be of clinical significance. These interactions should be investigated in patients with abnormal renal function.

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

1. 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. 2. 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. 3. 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. 4. 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 AUC(inf) 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. 5. 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.

Influence of tumor necrosis factor-alpha on the expression and function of P-glycoprotein in an immortalised rat brain capillary endothelial cell line, GPNT.

Drug cerebral pharmacokinetics may be altered in the case of inflammatory diseases. This may be due to a modification of drug transport through the blood-brain barrier, in particular through drug interaction with the membrane efflux transporter, P-glycoprotein. The objective of this study was to investigate the influence of the inflammatory cytokine, tumor necrosis factor (TNF)-alpha, on the functionality and expression of P-glycoprotein, and on mdr1a and mdr1b mRNA expression in immortalised rat brain endothelial cells, GPNT. Cells were treated with TNF-alpha for 4 days. Levels of mdr1a and mdr1b mRNAs were quantitated using real-time RT-PCR analysis and expression of P-glycoprotein was analyzed by Western blot. The functionality of P-glycoprotein was studied by following the accumulation of [3H]vinblastine in the cells without and with a pre-treatment with a P-glycoprotein inhibitor, GF120918. TNF-alpha increased the levels of mdr1a and mdr1b mRNAs while no effect was observed on protein expression. TNF-alpha increased [3H]vinblastine accumulation indicating a time and concentration-dependent decrease of P-glycoprotein activity. This effect was eliminated when the cells were pre-treated with GF120918. Our observation of a decrease in P-glycoprotein activity could suggest that in the case of inflammatory diseases, brain delivery of P-glycoprotein-dependent drugs can be enhanced.

Influence of the pro-inflammatory cytokines on P-glycoprotein expression and functionality.

PURPOSE: P-glycoprotein (P-gp) is involved in the transport of many drugs at different barriers with consequence in terms of drug distribution and elimination. The expression and activity of P-gp can be modulated by different factors and pathologies. The present article reviews the knowledge regarding the effect of pro-inflammatory cytokines (TNFalpha, IL-1beta, IL-6, IL-2, IFNgamma) on the expression and the functionality of P-gp at three major sites of drug absorption and disposition: the liver, the blood-brain barrier, and the intestine. METHODS: The various methods used to study the effect of pro-inflammatory cytokines include in vivo models (i.e. animals infected with Staphylococcus sp, animals injected with bacterial lipopolysaccharide or directly with cytokines, ...) and in vitro models (i.e. primary rat hepatocytes, human brain endothelial cells, ...). RESULTS: The data on P-gp expression and/or function may differ according to the compound used to induce inflammation. However, there is a general trend towards a decrease in both the expression of P-gp (mRNA and protein) and its functionality. Transcription factors and nuclear receptors are probably involved in this regulation. CONCLUSION: Cytokines may interfere with P-gp. Hence, in pathological conditions (inflammation, infection, ...), the expression and functionality of P-glycoprotein may be modulated with consequences for drug disposition and, consequently treatment efficacy.

Stereoselective distribution and stereoconversion of zopiclone enantiomers in plasma and brain tissues in rats.

Concentrations of (-)-zopiclone and (+)-zopiclone were determined in plasma and brain after oral administration, to investigate the stereoselectivity of distribution in rats. Zopiclone enantiomers were administered separately to rats and concentrations were determined by chiral HPLC in plasma and brain. In initial experiments, rats were treated with urethane before cannulation for blood sampling but as this drug modified zopiclone pharmacokinetics, it was not used in subsequent studies. This study showed that zopiclone pharmacokinetics after oral gavage in rats are stereoselective. After oral administration of (+)-zopiclone, no stereoconversion was observed in plasma. Conversely, after administration of (-)-zopiclone, both enantiomers were found in plasma and brain with (+)-zopiclone/(-)-zopiclone ratios of 1 and 8.4 in plasma and brain, respectively. Our findings suggest that zopiclone undergoes stereoconversion and that it is stereospecifically distributed to the brain.

Impact of cerebral malaria on brain distribution of mefloquine.

Cerebral malaria (CM) is the most severe complication of Plasmodium falciparum malaria. The aim of this study was to investigate the influence of CM on the cerebral uptake of mefloquine (MQ), in an experimental model of mice infected with Plasmodium berghei ANKA (PbA). Drug diffusion in brain is closely related to efflux pumps such as P-glycoprotein (P-gp/ABCB1/MDR1) and Breast Cancer Resistant Protein (BCRP/ABCG2), two major components of the blood-brain barrier (BBB) which can be modified by inflammation and/or infection. After a single IP dose, MQ concentrations were measured by liquid chromatography in blood and brains of mice infected with Plasmodium berghei ANKA and compared with that of non-infected mice. Our results show that MQ brain concentrations were decreased in CM mice versus healthy mice (0.77 versus 1.31 for brain/plasma concentrations). Although MQ is transported out of endothelial cells by P-glycoprotein, this result cannot be related to this transporter as we have previously shown that CM does not alter P-gp function (personal data). CM induces a reduction of MQ brain transport and, therefore, an increase of central toxicity due to MQ should not be expected during CM.

Efavirenz does not interact with the ABCB1 transporter at the blood-brain barrier.

PURPOSE: This work characterizes the interactions between efavirenz (EFV) and P-glycoprotein (P-gp/ABCB1) at the blood-brain barrier (BBB) and predicts the possible consequences on the brain uptake of coadministered P-gp substrates. METHODS: The uptake of EFV was measured in whole brains of rat and mdr1a-/- and mdr1a+/+ mice, and in GPNT cells (rat brain endothelial cell line) with and without P-gp inhibitors (PSC833, S9788, Quinidine). The effect of a single dose or multiple doses of EFV on the P-gp functionality was evaluated in vivo and in vitro by measuring the brain and cell uptake of digoxin, completed by the analysis of the P-gp expression at the rat BBB after repeated administrations of EFV. RESULTS: Inhibition of P-gp did not alter the uptake of EFV in rat brain and GPNT cells. The EFV brain/plasma ratio in mdr1a-/- mice, lacking the expression of P-gp, was not different from that in mdr1a+/+ mice. Moreover, a single dose of EFV did not modify the uptake of digoxin in rat brain and GPNT cells. Finally, the 3-day exposure of GPNT cells to EFV did not have any effect on the uptake of digoxin. Similarly, the 7-day treatment with EFV did not change the uptake of digoxin in rat brain nor the expression of P-gp at the BBB. CONCLUSION: EFV is strongly distributed in the brain, but is neither a substrate nor an inhibitor of the P-gp at the blood-brain barrier. On the other hand, EFV did not induce P-gp, allowing to sustain the brain accumulation of associated P-gp substrates such as protease inhibitors. These findings make EFV suitable for combinations circumventing the brain HIV-1 residency.

Transport of HIV protease inhibitors through the blood-brain barrier and interactions with the efflux proteins, P-glycoprotein and multidrug resistance proteins.

HIV protease inhibitors (HPIs) have limited penetration into the brain. This poor transport through the blood-brain barrier is mainly due to active efflux by proteins such as P-glycoprotein (P-gp) preventing drugs from clearing the brain of the virus. The present paper focuses on cerebral uptake of HPIs and interactions between HPIs and efflux proteins, either as substrates or modulators. Most of the studies described HPIs as P-gp substrates. Studies are more controversial when investigating HPIs as inhibitors of P-gp. HPIs seem to be able to inhibit efflux proteins of in vitro cell models but with limited consequences in vivo. Moreover, after repeated administrations of HPIs, most of them are also able to induce the expression and functionality of P-gp. For these reasons, certain combinations of HPIs may not efficiently increase brain uptake of HPIs as would combinations of more potent efflux inhibitors.

Effect of recombinant interleukin-2 pretreatment on oral and intravenous digoxin pharmacokinetics and P-glycoprotein activity in mice.

P-glycoprotein (P-gp) is an ATP-dependent efflux membrane transporter involved in many drug pharmacokinetics in humans. Decreasing its expression could enhance the bioavailability of substrates as digoxin. We have recently found that human recombinant interleukin-2 (rIL2) in vivo decreases P-gp expression in intestine and brain of mice and modifies oral digoxin pharmacokinetics. The aim of the study was to evaluate the involvement of bioavailability in the rIL2 pretreatment effect on digoxin pharmacokinetics by comparing oral and i.v. digoxin pharmacokinetics before and after rIL2 pretreatment (10 microg/kg). We also tried to show the possible effect of a low rIL2 dose (1 microg/kg) pretreatment on oral digoxin pharmacokinetics. First, adult Swiss mice received a single oral or i.v. dose of digoxin (0.03 mg/kg). Two weeks later, the same animals were treated by rIL2 i.p. twice a day (10 microg/kg) for 4 days and received digoxin again at day 5. As well, another group received oral digoxin (0.03 mg/kg) with a 1 microg/kg rIL2 pretreatment. Blood was collected after digoxin administration with and without rIL2 pretreatment. Digoxin pharmacokinetics were described by a one-compartment model. The 10 microg/kg rIL2 pretreatment did not modify i.v. digoxin pharmacokinetics, whereas oral digoxin pharmacokinetics were significantly modified by the 10 microg/kg rIL2 pretreatment and not by the 1 microg/kg rIL2 pretreatment. The decrease of P-gp activity, caused by rIL2 (10 microg/kg), increased digoxin bioavailability. An increase in exposure and intracellular level of drugs is expected from rIL2 pretreatment.

Effect of efflux inhibition on brain uptake of itraconazole in mice infected with Cryptococcus neoformans.

Itraconazole is a fungistatic agent that, although highly lipophilic, shows poor transport through the blood brain barrier that may be due to efflux proteins. The combined administration of an efflux inhibitor with itraconazole should increase cerebral itraconazole concentrations and therefore, improve the treatment of Cryptococcus neoformans meningitis with this antifungal agent. To test this hypothesis, we have studied the influence of murine cerebral infection with C. neoformans and the inhibition of efflux by intraperitoneal injection of a P-glycoprotein inhibitor, GF120918 [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-phenyl)9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide], on the pharmacokinetics of itraconazole in plasma and brain after a single intraperitoneal itraconazole injection. We also investigated the influence of efflux inhibition on the efficacy of repeated doses of itraconazole in this murine model. The results showed that in healthy and infected mice pretreated or not with GF120918, plasma itraconazole values of area under the curve (AUC) were similar. In contrast, cerebral values of AUC were higher in infected mice compared with healthy mice. Moreover, the pretreatment of infected mice with GF120918 significantly increased cerebral itraconazole values of area under the curve and decreased weight loss in the treatment with itraconazole of a cerebral infection with C. neoformans.

Glucocorticoid pharmacokinetics and growth retardation in children with renal transplants.

Long-term glucocorticoid treatment contributes to the growth retardation in children after renal transplantation. We investigated whether determination of prednisone (PN) and prednisolone (PL) in plasma and PN, PL, and 6-beta-hydroxyprednisolone (betaOH-PL) in urine could help to predict growth. PN and PL pharmacokinetics were studied in 36 children, from 5 to 15 years of age, receiving daily (D) or alternate-day (AD) oral PN treatment. Statural growth velocity was evaluated over a 1-year period. We compared three groups of children according to the growth kinetics during the study year (catch-up, stable, or decline) for clinical and pharmacokinetic parameters. A multiple linear regression analysis was performed in order to determine pharmacokinetic parameters able to explain height 1 year after inclusion. Height at the beginning of the study, creatinine clearance, and type of D or AD treatment explained 94.2% of height variance 1 year after inclusion. Only PL clearance was associated with growth evolution, but introduction of PL clearance in the multivariate model did not improve the variance of height accounted for by the previous model. We, therefore, do not recommend using glucocorticoid pharmacokinetics to predict growth retardation in children with renal transplantation.