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More accurate prediction of the extent of drug brain exposure in early drug discovery and understanding potential species differences could help to guide medicinal chemistry and avoid unnecessary animal studies. Hence, the aim of the current study was to validate the use of a P-gp transfected LLC-PK1 model to predict the unbound brain-to-plasma concentration ratio (Kpuu,brain) in rats and humans. MOCK-, Mdr1a- and MDR1-transfected LLC-PK1 monolayers were applied in a transwell setup to quantify the bidirectional transport for 12 specific P-gp substrates, 48 UCB drug discovery compounds, 11 compounds with reported rat in situ brain perfusion data and 6 compounds with reported human Kpuu,brain values. The in vitro transport data were introduced in a minimal PBPK model (SIVA®) to determine the transport parameters. These parameters were combined with the differences between in vitro and in vivo passive permeability as well as P-gp expression levels (as determined by LC-MS/MS), to predict the Kpuu,brain. A 10-fold difference between in vitro and in vivo passive permeability was observed. Incorporation of the differences between in vitro and in vivo passive permeability and P-gp expression levels resulted in an improved prediction of rat (AAFE 2.17) and human Kpuu,brain (AAFE 2.10). We have succesfully validated a methodology to use a P-gp overexpressing LLC-PK1 cell line to predict both rat and human Kpuu,brain by correcting for both passive permeability and P-gp expression levels.

With a long half-life, pharmacokinetic (PK) evaluation of monoclonal antibodies in rodents lasts multiple weeks during which the animals may grow significantly. We evaluated the impact of weight, age, and previous drug exposure on the PK of rituximab. Serum concentrations of rituximab were measured after intravenous and subcutaneous dosing in Sprague Dawley rats aged between 7 and 21 weeks and weighing between 200 and 600 g. The growth of rats during the study was incorporated into the model through the increase of the volumes of compartments in relation to the rats total body weight. The final model successfully captured all the data; and no difference was observed in the rituximab PK profiles between exposure naïve and redosed or young and older rats. Incorporating the rodent growth over the time course of the study into the PK model was shown to be important for providing a more physiological description of the disposition of rituximab, especially when young and rapidly growing animals are used. Redosing the same rats with monoclonal antibodies might be a viable strategy for reducing the use of laboratory animals in accordance with the 3R principles.

Diphenhydramine, a sedative histamine H1-receptor (H1R) antagonist, was evaluated as a probe to measure drug/H+-antiporter function at the blood–brain barrier. In situ brain perfusion experiments in mice and rats showed that diphenhydramine transport at the blood–brain barrier was saturable, following Michaelis–Menten kinetics with a Km = 2.99 mM and Vmax = 179.5 nmol s−1 g−1. In the pharmacological plasma concentration range the carrier-mediated component accounted for 77% of diphenhydramine influx while passive diffusion accounted for only 23%. [14C]Diphenhydramine blood–brain barrier transport was proton and clonidine sensitive but was influenced by neither tetraethylammonium, a MATE1 (SLC47A1), and OCT/OCTN (SLC22A1-5) modulator, nor P-gp/Bcrp (ABCB1a/1b/ABCG2) deficiency. Brain and plasma kinetics of [11C]diphenhydramine were measured by positron emission tomography imaging in rats. [11C]Diphenhydramine kinetics in different brain regions were not influenced by displacement with 1 mg kg−1 unlabeled diphenhydramine, indicating the specificity of the brain positron emission tomography signal for blood–brain barrier transport activity over binding to any central nervous system target in vivo. [11C]Diphenhydramine radiometabolites were not detected in the brain 15 min after injection, allowing for the reliable calculation of [11C]diphenhydramine brain uptake clearance (Clup = 0.99 ± 0.18 mL min−1 cm−3). Diphenhydramine is a selective and specific H+-antiporter substrate. [11C]Diphenhydramine positron emission tomography imaging offers a reliable and noninvasive method to evaluate H+-antiporter function at the blood–brain barrier.

Background and Purpose The respective impact and interplay between ABC (P-glycoprotein/P-gp/Abcb1a, BCRP/ABCG2, MRP/ABCC) and SLC transporter functions at the blood–brain barrier (BBB) and blood–retinal barriers (BRB) are incompletely understood. Experimental Approach We measured the initial cerebral and retinal distribution of selected ABC substrates by in situ carotid perfusion using P-gp/Bcrp knockout mice and chemical ABC/SLC modulation strategies. P-gp, Bcrp, Mrp1 and Mrp4 were studied by confocal retina imaging. Key Results Chemical or physical disruption of P-gp increased [3H]-verapamil transport by ~10-fold at the BBB and ~1.5-fold at the BRB. [3H]-Verapamil transport involved influx-mediated by an organic cation clonidine-sensitive/diphenhydramine-sensitive proton antiporter at both barriers; this effect was unmasked when P-gp was partially or fully inhibited/disrupted at the BBB. Studies of [3H]-mitoxantrone and [3H]-zidovudine transport suggested, respectively, that Bcrp efflux was less involved at the BRB than BBB, whereas Mrps were significantly and similarly involved at both barriers. Confocal imaging showed that P-gp and Bcrp were expressed in intra-retinal vessels (inner BRB/iBRB) but absent from the blood/basal membrane of cells of the retinal pigment epithelium (outer BRB/oBRB/RPE) where, in contrast, Mrp1 and Mrp4 were localized. Conclusions and Implications P-gp, Bcrp, Mrp1 and Mrp4 are differentially expressed at the outer and inner BRB, resulting in an altered ability to limit substrate distribution at the retina as compared with the BBB. [3H]-Verapamil distribution is not P-gp-specific and involves a proton antiporter at both the BBB and BRB. However, this transport is concealed by P-gp at the BBB, but not at the BRB, where P-gp activity is reduced.

Background and purpose An influx drug/proton antiporter of unknown structure has been functionally demonstrated at the blood-brain barrier. This transporter, which handles some psychoactive drugs like diphenhydramine, clonidine, oxycodone, nicotine and cocaine, could represent a new pharmacological target in drug addiction therapy. However, at present there are no known drugs/inhibitors that effectively inhibit/modulate this transporter in vivo. Experimental approach The FLAPpharm approach was used to establish a pharmacophore model for inhibitors of this transporter. The inhibitory potency of 44 selected compounds was determined against the specific substrate, [(3)H]-clonidine, in the human cerebral endothelial cell line hCMEC/D3 and ranked as good, medium, weak or non-inhibitor. Key results The pharmacophore model obtained was used as a template to screen xenobiotic and endogenous compounds from databases [Specs, Recon2, Human Metabolome Database (HMDB), human intestinal transporter database], and hypothetical candidates were tested in vitro to determine their inhibitory capacity with [(3)H]-clonidine. According to the transporter database, 80% of the proton antiporter inhibitor candidates could inhibit P-glycoprotein/MDR1/ABCB1 and specificity is improved by reducing inhibitor size/shape and increasing water solubility. Virtual screening results using HMDB and Recon2 for endogenous compounds appropriately scored tryptamine as an inhibitor. Conclusions and implications The pharmacophore model for the proton-antiporter inhibitors was a good predictor of known inhibitors and allowed us to identify new good inhibitors. This model marks a new step towards the discovery of this drug/proton antiporter and will be of great use for the discovery and design of potent inhibitors that could potentially help to assess and validate its pharmacological role in drug addiction in vivo.

Background and Purpose Transporters at the blood-retinal barrier (BRB), as at the blood–brain barrier (BBB), regulate the distribution of compounds into the neural parenchyma. However, the expression of BRB transporters and their quantitative impact in vivo are still poorly understood. Experimental Approach Clonidine and diphenhydramine are substrates of a novel BBB drug/proton-antiporter. We evaluated their transport at the BRB by in situ carotid perfusion in wild-type or knocked-out mice for Oct1-3 (Slc22a1-3). Key Results At pharmacological exposure levels, carrier-mediated BRB influx was 2 and 12 times greater than the passive diffusion rate for clonidine and diphenhydramine, respectively. Functional identification demonstrated the involvement of a high-capacity potassium- and sodium-independent proton-antiporter that shared the features of the previously characterized clonidine, diphenhydramine and cocaine BBB transporter. The functional characterization suggests that SLC transporters Oct1-3, Mate1 (Slc47a1) and Octn1-2 (Slc22a4-5) are not involved. Melanin/retinal toxic drugs like antimalarials (amodiaquine, quinine), quinidine and tricyclic antidepressants (imipramine) acted as inhibitors of this proton-antiporter. The endogenous indole derivative tryptamine inhibited the transporter, unlike 5-HT (serotonin), dopamine or L-DOPA. Trans-stimulation experiments with [3H]-clonidine at the BRB indicated that diphenhydramine, nicotine, oxycodone, naloxone, tramadol, 3,4-methylenedioxyamphetamine (MDMA, ecstasy), heroin, methadone and verapamil are common substrates. Conclusions and Implications A proton-antiporter is physiologically involved in the transport of clonidine and diphenhydramine and is quantitatively more important than their passive diffusion flux at the mouse BRB. The features of this molecularly unidentified transporter highlight its importance in regulating drug delivery at the retina and suggest that it has the capacity to handle several drugs.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor activated by a variety of widespread persistent environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). It can transactivate the expression of several target genes. Recently AhR transcripts were detected in isolated human brain microvessels and in the hCMEC/D3 human cerebral microvascular endothelial cell line, an in vitro model of the human cerebral endothelium. To date AhR implication in the co-regulation of ABCB1, ABCG2 and CYP1B1 at human cerebral endothelium has not been addressed. Here we investigated whether AhR could co-regulate ABCB1, ABCG2 and CYP1B1 expressions in the hCMEC/D3 cell line. Exposure to TCDD induced a concentration-dependent increase in CYP1B1 expression. We demonstrated AhR involvement in the TCDD-mediated increase in CYP1B1 expression by using small interfering RNA against AhR. Western blotting analysis also revealed an increase in CYP1B1 protein expression following TCDD exposure in hCMEC/D3. Regarding ABCB1 and ABCG2, exposure to TCDD had no effect on their protein expressions and functional activities. In conclusion our data indicated a differential modulation of CYP1B1 and ABCB1/ABCG2 expressions in hCMEC/D3 cells following TCDD exposure.

Nicotine, the main tobacco alkaloid leading to smoking dependence, rapidly crosses the blood-brain barrier (BBB) to become concentrated in the brain. Recently, it has been shown that nicotine interacts with some organic cation transporters (OCT), but their influence at the BBB has not yet been assessed in vivo. In this study, we characterized the transport of nicotine at the mouse luminal BBB by in situ brain perfusion. Its influx was saturable and followed the Michaelis-Menten kinetics (K(m)=2.60 mM, V(max)=37.60 nmol/s/g at pH 7.40). At its usual micromolar concentrations in the plasma, most (79%) of the net transport of nicotine at the BBB was carrier-mediated, while passive diffusion accounted for 21%. Studies on knockout mice showed that the OCT Oct1-3, P-gp, and Bcrp did not alter [(3)H]-nicotine transport at the BBB. Neither did inhibiting the transporters Mate1, Octn, or Pmat. The in vivo manipulation of intracellular and/or extracellular pH, the chemical inhibition profile, and the trans-stimulation experiments demonstrated that the nicotine transporter at the BBB shared the properties of the clonidine/proton antiporter. The molecular features of this proton-coupled antiporter have not yet been identified, but it also transports diphenhydramine and tramadol and helps nicotine cross the BBB at a faster rate and to a greater extent. The pharmacological inhibition of this nicotine/proton antiporter could represent a new strategy to reduce nicotine uptake by the brain and thus help curb addiction to smoking.

Diphenhydramine, a sedative histamine H1-receptor (H1R) antagonist, was evaluated as a probe to measure drug/H+-antiporter function at the blood–brain barrier. In situ brain perfusion experiments in mice and rats showed that diphenhydramine transport at the blood–brain barrier was saturable, following Michaelis–Menten kinetics with a Km = 2.99 mM and Vmax = 179.5 nmol s−1 g−1. In the pharmacological plasma concentration range the carrier-mediated component accounted for 77% of diphenhydramine influx while passive diffusion accounted for only 23%. [14C]Diphenhydramine blood–brain barrier transport was proton and clonidine sensitive but was influenced by neither tetraethylammonium, a MATE1 (SLC47A1), and OCT/OCTN (SLC22A1-5) modulator, nor P-gp/Bcrp (ABCB1a/1b/ABCG2) deficiency. Brain and plasma kinetics of [11C]diphenhydramine were measured by positron emission tomography imaging in rats. [11C]Diphenhydramine kinetics in different brain regions were not influenced by displacement with 1 mg kg−1 unlabeled diphenhydramine, indicating the specificity of the brain positron emission tomography signal for blood–brain barrier transport activity over binding to any central nervous system target in vivo. [11C]Diphenhydramine radiometabolites were not detected in the brain 15 min after injection, allowing for the reliable calculation of [11C]diphenhydramine brain uptake clearance (Clup = 0.99 ± 0.18 mL min−1 cm−3). Diphenhydramine is a selective and specific H+-antiporter substrate. [11C]Diphenhydramine positron emission tomography imaging offers a reliable and noninvasive method to evaluate H+-antiporter function at the blood–brain barrier.

International audience; In clinical practice, rifampicin exposure is estimated from its concentration in venous blood samples. In this study, we hypothesized that differences in rifampicin concentration may exist between arterial and venous plasma. An HPLC-UV method for determining rifampicin concentration in plasma using rifapentine as an internal standard was validated. The method, which requires a simple protein precipitation procedure as sample preparation, was performed to compare venous and arterial plasma kinetics after a single therapeutic dose of rifampicin (8.6 mg/kg i.v, infused over 30 min) in baboons (n = 3). The method was linear from 0.1 to 40 ␮g mL −1 and all validation parameters fulfilled the international requirements. In baboons, rifampicin concentration in arterial plasma was higher than in venous plasma. Arterial C max was 2.1 ± 0.2 fold higher than venous C max. The area under the curve (AUC) from 0 to 120 min was ∼80% higher in arterial plasma, indicating a significant arteriovenous concentration gradient in early rifampicin pharmacokinetics. Arterial and venous plasma concentrations obtained 6 h after rifampicin injection were not different. An important arteriovenous equilibration delay for rifampicin pharmacokinetics is reported. Determination in venous plasma concentrations may considerably underestimate rifampicin exposure to organs during the distribution phase.

Transporters at the blood-retinal barrier (BRB), as at the blood-brain barrier (BBB), regulate the distribution of compounds into the neural parenchyma. However, the expression of BRB transporters and their quantitative impact in vivo are still poorly understood.Clonidine and diphenhydramine are substrates of a novel BBB drug/proton-antiporter. We evaluated their transport at the BRB by in situ carotid perfusion in wild-type or knocked-out mice for Oct1-3 (Slc22a1-3).At pharmacological exposure levels, carrier-mediated BRB influx was 2 and 12 times greater than the passive diffusion rate for clonidine and diphenhydramine, respectively. Functional identification demonstrated the involvement of a high-capacity potassium- and sodium-independent proton-antiporter that shared the features of the previously characterized clonidine, diphenhydramine and cocaine BBB transporter. The functional characterization suggests that SLC transporters Oct1-3, Mate1 (Slc47a1) and Octn1-2 (Slc22a4-5) are not involved. Melanin/retinal toxic drugs like antimalarials (amodiaquine, quinine), quinidine and tricyclic antidepressants (imipramine) acted as inhibitors of this proton-antiporter. The endogenous indole derivative tryptamine inhibited the transporter, unlike 5-HT (serotonin), dopamine or L-DOPA. Trans-stimulation experiments with [(3) H]-clonidine at the BRB indicated that diphenhydramine, nicotine, oxycodone, naloxone, tramadol, 3,4-methylenedioxyamphetamine (MDMA, ecstasy), heroin, methadone and verapamil are common substrates.A proton-antiporter is physiologically involved in the transport of clonidine and diphenhydramine and is quantitatively more important than their passive diffusion flux at the mouse BRB. The features of this molecularly unidentified transporter highlight its importance in regulating drug delivery at the retina and suggest that it has the capacity to handle several drugs.

International audience; Background: The rate of entry of cocaine into the brain is a critical factor that influences neuronal plasticity and the development of cocaine addiction. Until now, passive diffusion has been considered the unique mechanism known by which cocaine crosses the blood-brain barrier. Methods: We reassessed mechanisms of transport of cocaine at the blood-brain barrier using a human cerebral capillary endothelial cell line (hCMEC/D3) and in situ mouse carotid perfusion. Results: Both in vivo and in vitro cocaine transport studies demonstrated the coexistence of a carrier-mediated process with passive diffusion. At pharmacological exposure level, passive diffusion of cocaine accounted for only 22.5% of the total cocaine influx in mice and 5.9% in hCMEC/D3 cells, whereas the carrier-mediated influx rate was 3.4 times greater than its passive diffusion rate in vivo. The functional identification of this carrier-mediated transport demonstrated the involvement of a proton antiporter that shared the properties of the previously characterized clonidine and nicotine transporter. The functionnal characterization suggests that the solute carrier (SLC) transporters Oct (Slc22a1-3), Mate (Slc47a1) and Octn (Slc22a4-5) are not involved in the cocaine transport in vivo and in vitro. Diphenhydramine, heroin, tramadol, cocaethylene, and norcocaine all strongly inhibited cocaine transport, unlike benzoylecgonine. Trans-stimulation studies indicated that diphenhydramine, nicotine, 3,4-methylenedioxyamphetamine (ecstasy) and the cathinone compound 3,4-methylenedioxypyrovalerone (MDPV) were also substrates of the cocaine transporter. Conclusions: Cocaine transport at the BBB involves a proton-antiporter flux that is quantitatively much more important than its passive diffusion. The molecular identification and characterization of this transporter will provide new tools to understand its role in addictive mechanisms.

Transport across the BBB is a determinant of the rate and extent of drug distribution in the brain. Heroin exerts its effects through its principal metabolites 6-monoacetyl-morphine (6-MAM) and morphine. Morphine is a known substrate of P-glycoprotein (P-gp) at the blood-brain-barrier (BBB) however, little is known about the interaction of heroin and 6-MAM with P-gp. The objective of this paper is to study the role of the P-gp-mediated efflux at the BBB in the behavioral and molecular effects of heroin and morphine. The transport rates of heroin and its main metabolites, at the BBB, were measured in mice by in situ brain perfusion. We then examined the effect of inhibition of P-gp on the acute nociception, locomotor activity, and gene expression modulations induced by heroin and morphine. The effect of P-gp inhibition during the acquisition of morphine-induced place preference was also studied. Inhibition of P-gp significantly increased the uptake of morphine but not that of heroin nor 6-MAM. Inhibition of P-gp significantly increased morphine-induced acute analgesia and locomotor activity but did not affect the behavioral effects of heroin; in addition, acute transcriptional responses to morphine were selectively modulated in the nucleus accumbens. Increasing morphine uptake by the brain significantly increased its reinforcing properties in the place preference paradigm. The present study demonstrated that acute inhibition of P-gp not only modulates morphine-induced behavioral effects but also its transcriptional effects and reinforcing properties. This suggests that, in the case of morphine, transport across the BBB is critical for the development of dependence.

Effects of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), natural ligand of the VDR, on the fates of adefovir dipivoxil (P-gp substrate) and its metabolites, mono(POM)-PMEA and adefovir (MRP4 substrate), were investigated in Caco-2 cells. After 1,25(OH)2D3-treatment, higher apical efflux of adefovir was observed after a 60 min incubation of adefovir divipoxil. Changes in these washout studies were predicted by a catenary model for the Caco-2 monolayer that described a higher MRP4 activity with 1,25(OH)2D3 treatment, as confirmed by Western blotting. Moreover, 1,25(OH)2D3 treatment (100 nM for 3 days) resulted in increased basolateral (B) to apical (A) (B-to-A) transport of adefovir dipivoxil but an unchanged A-to-B flux, rendering an elevated efflux ratio (EfR) (from 1.97 to 3.19). The EfR values in control and 1,25(OH)2D3-treated groups in these transport studies were reduced to 1.32 and 1.57, respectively, in the presence of verapamil (50 μM), the P-gp inhibitor. The B-to-A transport of the metabolite, adefovir, was increased in 1,25(OH)2D3-treated cells in the presence of verapamil, whereas the A-to-B and B-to-A transport of mono(POM)-PMEA remained unchanged. But the verapamil and 1,25(OH)2D3 treatments failed to alter rates of sequential metabolism of adefovir dipivoxil in cell lysate. The composite data established that 1,25(OH)2D3 treatment increased both P-gp and MRP4 transport activities without affecting the metabolism of adefovir dipivoxil by esterases. Moreover, an asymmetric appearance of metabolites, being higher with apical application, was observed. According to the catenary model, the asymmetry is suggestive that esterases are predominantly localized on the apical membrane and within the cell.