12 research outputs found
Functional and molecular identification of a transporter of psychotropic and drugs of abuse
Le systĂšme nerveux central est un organe privilĂ©giĂ© et protĂ©gĂ©, notamment grĂące Ă lâexistence des barriĂšres histologiques entre le sang et les tissus nerveux. La barriĂšre-hĂ©mato encĂ©phalique (BHE) et la barriĂšre hĂ©mato-rĂ©tinienne (BHR) sĂ©parent respectivement le parenchyme cĂ©rĂ©bral et la rĂ©tine des composĂ©s contenus dans lâespace vasculaire, grĂące Ă lâexpression de jonctions serrĂ©es et de transporteurs membranaires permettant une rĂ©gulation spĂ©cifique des Ă©changes entre le sang et le parenchyme nerveux. Ce travail a portĂ© sur lâĂ©tude dâun nouveau transporteur de cations organiques mis en Ă©vidence fonctionnellement Ă la BHE de la souris. Ce transporteur appartenant trĂšs probablement Ă la superfamille des solute carrier (SLC), fonctionne comme un antiport proton. Actuellement, sa prĂ©sence ne peut ĂȘtre dĂ©montrĂ©e que de façon fonctionnelle car son identitĂ© molĂ©culaire est encore inconnue. Cet antiport proton constitue un nouvel acteur de la permĂ©abilitĂ© cĂ©rĂ©brale et ouvre une nouvelle voie dâaccĂšs au cerveau. Nous nous sommes tout dâabord attachĂ©s Ă approfondir les connaissances fonctionnelles de ce transporteur en Ă©tudiant de nouveaux substrats et tissus dâexpression. Le transport cĂ©rĂ©bral de psychotropes a Ă©tĂ© Ă©tudiĂ© in vivo par la technique de perfusion carotidienne in situ chez la souris et in vitro grĂące Ă une lignĂ©e de cellules endothĂ©liales cĂ©rĂ©brales humaines immortalisĂ©es (hCMEC/D3). Nous avons dĂ©montrĂ© que la haute permĂ©abilitĂ© cĂ©rĂ©brale de la cocaĂŻne fait intervenir Ă la fois une diffusion passive et surtout une diffusion mĂ©diĂ©e par un antiport proton. La vitesse dâentrĂ©e des substances dâabus dans le cerveau est associĂ©e Ă un plus fort risque dâaddiction et fait de ce transporteur un nouvel acteur critique de la rĂ©gulation du passage cĂ©rĂ©bral. En effet, dâautres substances comme la nicotine et certaines amphĂ©tamines comme le MDPV et l'ecstasy sont Ă©galement des substrats de cet antiport. Ce transporteur apparaĂźt comme une cible pharmacologique potentielle dans la prise en charge de toxicomanies. MalgrĂ© la diversitĂ© chimique et pharmacologique dâinteractions des composĂ©s avec cet antiport, les concentrations nĂ©cessaires pour lâinhiber dĂ©passent celles retrouvĂ©es dans le sang. Pour aider lâidentification dâinhibiteurs sĂ©lectifs et efficaces nous avons dĂ©veloppĂ© un modĂšle pharmacophorique dâinhibiteurs du transporteur Ă partir de donnĂ©es gĂ©nĂ©rĂ©es in vitro et de lâapproche FLAPpharm. Ce modĂšle semble prĂ©dictif de nouveaux composĂ©s pouvant constituer de meilleurs inhibiteurs de ce transporteur. LâĂ©tude des Ă©changes in vivo au niveau du tissu nerveux nous a menĂ©s Ă Ă©tudier lâimpact de transporteurs ABC et de lâantiport-proton au niveau cĂ©rĂ©bral et rĂ©tinien Ă lâaide de substances spĂ©cifiques ou de substrats mixtes comme le vĂ©rapamil. Lâantiport proton est fonctionnel au niveau de la BHR et transporte notamment la clonidine, le DPH et le vĂ©rapamil. Cependant, dans le cas dâun substrat mixte P-gp et SLC (ex : vĂ©rapamil), ce transport dâinflux nâest visible Ă la BHE que lorsque la P-gp est neutralisĂ©e. Au contraire, Ă la BHR lâinflux liĂ© Ă cet SLC est visible naturellement. Lâimpact de la P-gp Ă la BHR Ă©tant 6.3-fois plus faible ce processus est probablement moins masquĂ©. Cette Ă©tude illustre la difficultĂ© actuelle de prĂ©dire lâimpact fonctionnel dâun transporteur pour des substrats multi-spĂ©cifiques et lâexistence dâune priorisation du transport. Enfin, nous avons essayĂ© dâidentifier lâantiport proton au niveau molĂ©culaire par une mĂ©thode de photo-activation Ă lâaide dâun composĂ© adaptĂ©. Cette mĂ©thode sâest avĂ©rĂ©e efficace pour fixer une molĂ©cule sur le transporteur, permettant par la suite de lâisoler plus facilement. En conclusion, ce travail a permis de mettre en Ă©vidence lâimportance de lâantiport proton dans la distribution cĂ©rĂ©brale de psychotropes et dâouvrir de nouvelles perspectives dans lâaddiction et la comprĂ©hension du transport de substrats multi-spĂ©cifiques.The central nervous system is a privilege organ protected by histological barriers between the blood and the nervous tissue. The blood-brain barrier (BBB) and the blood-retinal barrier (BRB) separate cerebral parenchyma and retina from the circulating blood and both express tight junctions and membrane transporters, allowing a precise regulation of the exchanges between the blood and nervous tissues. We studied a new cationic transporter functionally evidenced at the mouse BBB. This molecularly unknown transporter belong to the solute carrier super family (SLC) and is a proton antiporter. It could constitute a new actor in the cerebral permeability and may be a new brain access pathway. First, we worked on the functional identification studying new substrates and new localization. Psychotropic brain transport was studied in vivo by brain in situ perfusion on mouse and in vitro with human immortalized endothelial cells (hCMEC/D3). We showed that cocaine brain entry depends on passive diffusion but also mainly on a proton antiporter. Brain entry rate of drugs of abuse is associated with modulation of addiction liability, making this transporter a new component of brain entry of cocaine, and also nicotine and some amphetamines such as ecstasy and MDPV. This proton antiporter appears to be a new potential target in addiction. Various chemical entities interact with this transporter; however concentrations used to inhibit the transporter are much higher than the one possibly found in the blood. In order to help find or design new selective and potent inhibitors, we developed a pharmacophore model of the proton antiporter inhibitors using in vitro data and the FLAPpharm approach. The model predicts well new possible inhibitors of this transporter. We also studied the impact of the ABC transporters and the proton antiporter at the BBB and the BRB using specific or multi-specific substrates such as verapamil. The proton antiporter is functionally expressed at the BRB and transports clonidine, DPH and verapamil. However, for the multi-specific (P-gp and SLC) compound verapamil, influx transport by the proton antiporter is visible at the BBB only when P-gp efflux is neutralized. On the contrary, at the BRB, the proton antiporter influx is always visible. This is certainly due to the lower impact (by 6.3 fold) of P-gp at the BRB compared to the BBB. These results show the difficulty to predict the functional impact of a transporter for multi-specific compounds and a probable transport prioritization. Finally we worked on the molecular identification of the proton antiporter using a photolabeling method. This work evidenced the importance of the proton antiporter in the brain distribution of psychotropic and drugs of abuse and opened toward new perspectives in addiction and transport comprehension
Evaluation de l'activité et de l'expression des transporteurs hépatiques OATP1B1 et OATP1B3 (application de facteurs d'extrapolation aux simulations PBPK)
PARIS-BIUP (751062107) / SudocSudocFranceF
Pharmacophore-Based Discovery of Substrates of a Novel Drug/Proton-Antiporter in the Human Brain Endothelial hCMEC/D3 Cell Line
A drug/proton-antiporter, whose the molecular structure is still unknown, was previously evidenced at the blood-brain barrier (BBB) by functional experiments. The computational method could help in the identification of substrates of this solute carrier (SLC) transporter. Two pharmacophore models for substrates of this transporter using the FLAPpharm approach were developed. The trans-stimulation potency of 40 selected compounds for already known specific substrates ([3H]-clonidine) were determined and compared in the human brain endothelial cell line hCMEC/D3. Results. The two pharmacophore models obtained were used as templates to screen xenobiotic and endogenous compounds from four databases (e.g., Specs), and 45 hypothetical new candidates were tested to determine their substrate capacity. Psychoactive drugs such as antidepressants (e.g., imipramine, desipramine), antipsychotics/neuroleptics such as phenothiazine derivatives (chlorpromazine), sedatives anti-histamine-H1 drugs (promazine, promethazine, triprolidine, pheniramine), opiates/opioids (e.g., hydrocodone), trihexyphenidyl and sibutramine were correctly predicted as proton-antiporter substrates. The best performing pharmacophore model for the proton-antiporter substrates appeared as a good predictor of known substrates and allowed the identification of new substrate compounds. This model marks a new step in the characterization of this drug/proton-antiporter and will be of great use in uncovering its substrates and designing chemical entities with an improved influx capability to cross the BBB
Validation of a simple HPLC-UV method for rifampicin determination in plasma: Application to the study of rifampicin arteriovenous concentration gradient
International audienceIn 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 âźg mL â1 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 âŒ80% 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
Diphenhydramine as a selective probe to study H + -antiporter function at the bloodâbrain barrier: Application to [ 11 C]diphenhydramine positron emission tomography imaging
International audienceDiphenhydramine, a sedative histamine H 1 -receptor (H 1 R) antagonist, was evaluated as a probe to measure drug/H + -antiporter function at the bloodâbrain barrier. In situ brain perfusion experiments in mice and rats showed that diphenhydramine transport at the bloodâbrain barrier was saturable, following MichaelisâMenten kinetics with a K m =â2.99âmM and V max =â179.5ânmolâs â1 g â1 . In the pharmacological plasma concentration range the carrier-mediated component accounted for 77% of diphenhydramine influx while passive diffusion accounted for only 23%. [ 14 C]Diphenhydramine bloodâbrain 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 (ABCB 1a/1b /ABCG2) deficiency. Brain and plasma kinetics of [ 11 C]diphenhydramine were measured by positron emission tomography imaging in rats. [ 11 C]Diphenhydramine kinetics in different brain regions were not influenced by displacement with 1âmgâkg â1 unlabeled diphenhydramine, indicating the specificity of the brain positron emission tomography signal for bloodâbrain barrier transport activity over binding to any central nervous system target in vivo. [ 11 C]Diphenhydramine radiometabolites were not detected in the brain 15âmin after injection, allowing for the reliable calculation of [ 11 C]diphenhydramine brain uptake clearance (Cl up =â0.99â±â0.18âmLâmin â1 cm â3 ). Diphenhydramine is a selective and specific H + -antiporter substrate. [ 11 C]Diphenhydramine positron emission tomography imaging offers a reliable and noninvasive method to evaluate H + -antiporter function at the bloodâbrain barrier
Carrier-Mediated Cocaine Transport at the Blood-Brain Barrier as a Putative Mechanism in Addiction Liability
International audienceBackground: 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
Impact of P-glycoprotein at the blood-brain barrier on the uptake of heroin and its main metabolites: behavioral effects and consequences on the transcriptional responses and reinforcing properties
International audienceTransport 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