Functional and targeted proteomics characterization of a human primary endothelial cell model of the blood-brain barrier (BBB) for drug permeability studies.

The blood-brain barrier (BBB) protects the brain from toxins but hinders the penetration of neurotherapeutic drugs. Therefore, the blood-to-brain permeability of chemotherapeutics must be carefully evaluated. Here, we aimed to establish a workflow to generate primary cultures of human brain microvascular endothelial cells (BMVECs) to study drug brain permeability and bioavailability. Furthermore, we characterized and validated this BBB model in terms of quantitative expression of junction and drug-transport proteins, and drug permeability. We isolated brain microvessels (MVs) and cultured BMVECs from glioma patient biopsies. Then, we employed targeted LC-MS proteomics for absolute protein quantification and immunostaining to characterize protein localization and radiolabeled drugs to predict drug behavior at the Human BBB. The abundance levels of ABC transporters, junction proteins, and cell markers in the cultured BMVECs were similar to the MVs and correctly localized to the cell membrane. Permeability values (entrance and exit) and efflux ratios tested in vitro using the primary BMVECs were within the expected in vivo values. They correctly reflected the transport mechanism for 20 drugs (carbamazepine, diazepam, imipramine, ketoprofen, paracetamol, propranolol, sulfasalazine, terbutaline, warfarin, cimetidine, ciprofloxacin, digoxin, indinavir, methotrexate, ofloxacin, azidothymidine (AZT), indomethacin, verapamil, quinidine, and prazosin). We established a human primary in vitro model suitable for studying blood-to-brain drug permeability with a characterized quantitative abundance of transport and junction proteins, and drug permeability profiles, mimicking the human BBB. Our results indicate that this approach could be employed to generate patient-specific BMVEC cultures to evaluate BBB drug permeability and develop personalized therapeutic strategies.

Brain Distribution of Drugs: Brain Morphology, Delivery Routes, and Species Differences.

Neuropharmacokinetics considers cerebral drug distribution as a critical process for central nervous system drug action as well as for drug penetration through the CNS barriers. Brain distribution of small molecules obeys classical rules of drug partition, permeability, binding to fluid proteins or tissue components, and tissue perfusion. The biodistribution of all drugs, including both small molecules and biologics, may also be influenced by specific brain properties related to brain anatomy and physiological barriers, fluid dynamics, and cellular and biochemical composition, each of which can exhibit significant interspecies differences. All of these properties contribute to select optimal dosing paradigms and routes of drug delivery to reach brain targets for classical small molecule drugs as well as for biologics. The importance of these properties for brain delivery and exposure also highlights the need for efficient new analytical technologies to more comprehensively investigate drug distribution in the CNS, a complex multi-compartmentalized organ system.

Is Lockdown Effective in Limiting SARS-CoV-2 Epidemic Progression?-a Cross-Country Comparative Evaluation Using Epidemiokinetic Tools.

BACKGROUND: To date, the risk/benefit balance of lockdown in controlling severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) epidemic is controversial. OBJECTIVE: We aimed to investigate the effectiveness of lockdown on SARS-CoV-2 epidemic progression in nine different countries (New Zealand, France, Spain, Germany, the Netherlands, Italy, the UK, Sweden, and the USA). DESIGN: We conducted a cross-country comparative evaluation using a susceptible-infected-recovered (SIR)-based model completed with pharmacokinetic approaches. MAIN MEASURES: The rate of new daily SARS-CoV-2 cases in the nine countries was calculated from the World Health Organization's published data. Using a SIR-based model, we determined the infection (ß) and recovery (γ) rate constants; their corresponding half-lives (t1/2ß and t1/2γ); the basic reproduction numbers (R0 as ß/γ); the rates of susceptible S(t), infected I(t), and recovered R(t) compartments; and the effectiveness of lockdown. Since this approach requires the epidemic termination to build the (I) compartment, we determined S(t) at an early epidemic stage using simple linear regressions. KEY RESULTS: In New Zealand, France, Spain, Germany, the Netherlands, Italy, and the UK, early-onset stay-at-home orders and restrictions followed by gradual deconfinement allowed rapid reduction in SARS-CoV-2-infected individuals (t1/2ß ≤ 14 days) with R0 ≤ 1.5 and rapid recovery (t1/2γ ≤ 18 days). By contrast, in Sweden (no lockdown) and the USA (heterogeneous state-dependent lockdown followed by abrupt deconfinement scenarios), a prolonged plateau of SARS-CoV-2-infected individuals (terminal t1/2ß of 23 and 40 days, respectively) with elevated R0 (4.9 and 4.4, respectively) and non-ending recovery (terminal t1/2γ of 112 and 179 days, respectively) was observed. CONCLUSIONS: Early-onset lockdown with gradual deconfinement allowed shortening the SARS-CoV-2 epidemic and reducing contaminations. Lockdown should be considered as an effective public health intervention to halt epidemic progression.

Is Curfew Effective in Limiting SARS-CoV-2 Progression? An Evaluation in France Based on Epidemiokinetic Analyses.

BACKGROUND: Since late summer 2020, the French authorities implemented a curfew/lightened lockdown-alternating strategy instead of strict lockdown, to improve acceptability and limit socioeconomic consequences. However, data on curfew-related efficacy to control the epidemic are scarce. OBJECTIVE: To investigate the effects on COVID-19 spread in France of curfew combined to local and/or nationwide restrictions from late summer 2020 to mid-February 2021. DESIGN: We conducted a comparative evaluation using a susceptible-infected-recovered (SIR)-based model completed with epidemiokinetic tools. MAIN MEASURES: We analyzed the time-course of epidemic progression rate under curfew in French Guyana and five metropolitan regions where additional restrictions were implemented at different times. Using linear regressions of the decay/increase rates in daily contaminations, we calculated the epidemic regression half-lives (t1/2ß) for each identified period. KEY RESULTS: In French Guyana, two decay periods with rapid regression (t1/2ß of ~10 days) were observed under curfew, with slowing (t1/2ß of ~43 days) when curfew was lightened. During the 2-week pre-lockdown curfew (2020/10/17-2020/11/02) in Provence-Alpes-Côte-d'Azur, Auvergne-Rhône-Alpes, and Ile-de-France, the epidemic progression was unchanged. During the post-lockdown curfew (2020/12/15-2020/02/14), the epidemic slowly regressed in Grand-Est (t1/2ß of ~37 days), whereas its progression rate plateaued in Auvergne-Rhône-Alpes and increased immediately in Provence-Alpes-Côte-d'Azur, Ile-de-France, and Nouvelle-Aquitaine, whatever the curfew starting time was (06:00 or 08:00 pm). Interestingly, a delayed slow decay (17 days < t1/2ß < 64 days) occurred under curfew in all regions except Ile-de-France. CONCLUSIONS: Curfew allowed the temporary control of SARS-CoV-2 epidemic, however variably in the French regions, without preventing lockdown necessity. To accelerate the epidemic regression such as observed in French Guyana, curfew should be implemented timely with additional restrictions.

Targeted unlabeled multiple reaction monitoring analysis of cell markers for the study of sample heterogeneity in isolated rat brain cortical microvessels.

Liquid chromatography coupled to tandem mass spectrometry-based targeted absolute protein quantification (in fmol of the analyte protein per µg of total protein) is employed for the molecular characterization of the blood-brain barrier using isolated brain microvessels. Nevertheless, the heterogeneity of the sample regarding the levels of different cells co-isolated within the microvessels and bovine serum albumin (BSA) contamination (from buffers) are not always evaluated. We developed an unlabeled targeted liquid chromatography coupled to tandem mass spectrometry method to survey the levels of endothelial cells (ECs), astrocytes, and pericytes, as well as BSA contaminant in rat cortical microvessels. Peptide peak identities were evaluated using a spectral library and chromatographic parameters. Sprague-Dawley rat microvessels obtained on three different days were analyzed with this method complemented by an absolute quantification multiple reaction monitoring method for transporter proteins P-gp, Bcrp, and Na+ /K+ ATPase pump using stable isotope labeled peptides as internal standard. Inter-day differences in the cell markers and BSA contamination were observed. Levels of cell markers correlated positively between each other. Then, the correlation between cell marker proteins and transporter proteins was evaluated to choose the best EC marker protein for protein quantification normalization. The membrane protein Pecam-1 showed a very high correlation with the EC-specific transporter P-gp (Pearson product-moment correlation coefficient (r) > 0.89) and moderate to high with Bcrp (r ≥ 0.77), that can be found also in pericytes and astrocytes. Therefore, Pecam-1 was selected as a marker for the normalization of the quantification of the proteins of endothelial cells.

The effect of morbid obesity on morphine glucuronidation.

The purpose of the present work was to study the change in morphine metabolic ratio in obese subjects before and after Roux-en-Y Gastric Bypass (RYGB) and to identify clinical and/or biological factors associated with this change. The pharmacokinetics (PK) of oral morphine (30mg), morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) was performed in patients before (n=25; mean BMI=43.2 (35.4-61.9)kg/m2), 7-15days (n=16) and 6 months after RYGB (n=19; mean BMI=32.3 (25.4-46.0)kg/m2). Morphine Cmax and AUC0-inf were significantly increased and morphine Tmax significantly shortened at 6 months after RYGB compared with preoperative data, indicating an important increase in the rate and extent of morphine absorption. The morphine metabolic ratio 0-inf M3G+M6G/Morphine, decreased significantly from the preoperative to 6 months postoperative period with an average of -26% (range -74%; +21%; p=0.004), but not in the immediate post-operative period. The change in morphine metabolic ratio was associated with a change in BMI, fat mass in kg, and triglyceride levels (rho=0.5, p≤0.04). The degree of change in several markers of low-grade inflammation, or the level of liver steatosis and fibrosis before surgery, was not associated with the change in morphine metabolic ratios. Our findings indicate that RYGB-induced weight loss significantly decreases morphine metabolic ratio, arguing for an effect of morbid obesity on glucuronidation. With glucuronide exposure at 6 months similar to preoperative values, a higher morphine AUC0-inf should encourage reducing morphine dosage in patients undergoing RYGB and chronically receiving immediate-release oral morphine.

Oral Morphine Pharmacokinetic in Obesity: The Role of P-Glycoprotein, MRP2, MRP3, UGT2B7, and CYP3A4 Jejunal Contents and Obesity-Associated Biomarkers.

The objective of our work was to study the association between the jejunal expression levels of P-gp, MRP2, MRP3, UGT2B7, CYP3A4, the ABCB1 c.3435C > T polymorphism, and several obesity-associated biomarkers, as well as oral morphine and glucuronides pharmacokinetics in a population of morbidly obese subjects. The pharmacokinetics of oral morphine (30 mg) and its glucuronides was performed in obese patients candidate to bariatric surgery. A fragment of jejunal mucosa was preserved during surgery. Subjects were genotyped for the ABCB1 single nucleotide polymorphism (SNP) c.3435C > T. The subjects were 6 males and 23 females, with a mean body mass index of 44.8 (35.4-61.9) kg/m(2). The metabolic ratios AUC0-inf M3G/morphine and AUC0-inf M6G/morphine were highly correlated (rs = 0.8, p < 0.0001) and were 73.2 ± 24.6 (34.7-137.7) and 10.9 ± 4.1 (3.8-20.6). The pharmacokinetic parameters of morphine and its glucuronides were not associated with the jejunal contents of P-gp, CYP3A4, MRP2, and MRP3. The jejunal content of UGT2B7 was positively associated with morphine AUC0-inf (rs = 0.4, p = 0.03). Adiponectin was inversely correlated with morphine Cmax (rs = -0.44, p = 0.03). None of the factors studied was associated with morphine metabolic ratios. The interindividual variability in the jejunal content of drug transporters and metabolizing enzymes, the ABCB1 gene polymorphism, and the low-grade inflammation did not explain the variability in morphine and glucuronide exposure. High morphine metabolic ratio argued for an increased morphine glucuronidation in morbidly obese patients.

Quantitative Atlas of Cytochrome P450, UDP-Glucuronosyltransferase, and Transporter Proteins in Jejunum of Morbidly Obese Subjects.

Protein expression levels of drug-metabolizing enzymes and transporters in human jejunal tissues excised from morbidly obese subjects during gastric bypass surgery were evaluated using quantitative targeted absolute proteomics. Protein expression levels of 15 cytochrome P450 (CYP) enzymes, 10 UDP-glucuronosyltransferase (UGT) enzymes, and NADPH-P450 reductase (P450R) in microsomal fractions from 28 subjects and 49 transporters in plasma membrane fractions from 24 of the same subjects were determined using liquid chromatography-tandem mass spectrometry. Based on average values, UGT1A1, UGT2B15, UGT2B17, SGLT1, and GLUT2 exhibited high expression levels (over 10 fmol/µg protein), though UGT2B15 expression was detected at a high level in only one subject. CYP2C9, CYP2D6, CYP3A5, UGT1A6, P450R, ABCG2, GLUT5, PEPT1, MCT1, 4F2 cell-surface antigen heavy chain (4F2hc), LAT2, OSTα, and OSTß showed intermediate levels (1-10 fmol/µg protein), and CYP1A1, CYP1A2, CYP1B1, CYP2C18, CYP2C19, CYP2J2, CYP3A7, CYP4A11, CYP51A1, UGT1A3, UGT1A4, UGT1A8, UGT2B4, ABCC1, ABCC4, ABCC5, ABCC6, ABCG8, TAUT, OATP2A1, OATP2B1, OATP3A1, OATP4A1, OCTN1, CNT2, PCFT, MCT4, GLUT4, and SLC22A18 showed low levels (less than 1 fmol/µg protein). The greatest interindividual difference (364-fold) was detected for UGT2B17. However, differences in expression levels of other quantified UGTs (except UGT2B15 and UGT2B17), CYPs (except CYP1A1 and CYP3A5), and P450R, and all quantified transporters, were within 10-fold. Expression levels of CYP1A2 and GLUT4 were significantly correlated with body-mass index. The levels of 4F2hc showed significant gender differences. Smokers showed increased levels of UGT1A1 and UGT1A3. These findings provide a basis for understanding the changes in molecular mechanisms of jejunal metabolism and transport, as well as their interindividual variability, in morbidly obese patients.

Hypoxia interferes with aryl hydrocarbon receptor pathway in hCMEC/D3 human cerebral microvascular endothelial cells.

The expression of aryl hydrocarbon receptor (AhR) transcription factor was detected at transcript level in freshly isolated human brain microvessels and in the hCMEC/D3 human cerebral microvascular endothelial cell line. Recent studies have demonstrated that AhR pathway is able to crosstalk with other pathways such as hypoxia signaling pathway. Therefore, we used the hCMEC/D3 cell line to investigate the potential crosstalk between AhR and hypoxia signaling pathways. First, we performed two different hypoxia-like procedures in hCMEC/D3 cells; namely, exposition of cells to 150 µM deferoxamine or to glucose and oxygen deprivation for 6 h. These two procedures led to hypoxia-inducible factor (HIF)-1α and HIF-2α proteins accumulation together with a significant induction of the two well-known hypoxia-inducible genes VEGF and GLUT-1. Both HIF-1α and -2α functionally mediated hypoxia response in the hCMEC/D3 cells. Then, we observed that a 6 h exposure to 25 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin, a strong AhR ligand, up-regulated CYP1A1 and CYP1B1 expression, and that this effect was AhR dependent. Regarding AhR and hypoxia crosstalk, our experiments revealed that an asymmetric interference between these two pathways effectively occurred in hCMEC/D3 cells: hypoxia pathway interfered with AhR signaling but not the other way around. We studied the putative crosstalk of AhR and hypoxia pathways in hCMEC/D3 human cerebral microvascular endothelial cells. While hypoxia decreased the expression of the two AhR target genes CYP1A1 and CYP1B1, AhR activation results in no change in hypoxia target gene expression. This is the first sign of AhR and hypoxia pathway crosstalk in an in vitro model of the human cerebral endothelium.

Methadone dose in heroin-dependent patients: role of clinical factors, comedications, genetic polymorphisms and enzyme activity.

AIMS: Methadone is characterized by wide intersubject variability regarding the dose needed to obtain full therapeutic response. We assessed the influence of sociodemographic, ethnic, clinical, metabolic and genotypic variables on methadone maintenance dose requirement in opioid-dependent responder patients. METHODS: Eighty-one stable patients (60 men and 21 women, 43.7 ± 8.1 years old, 63.1 ± 50.9 mg day(-1) methadone), divided into quartiles with respect to the median daily dose, were enrolled and underwent clinical examination, treatment history and determination of liver/intestinal cytochrome P450 (CYP) 3A4 activity measured by the midazolam test, R,S-methadone trough concentration and clinically significant polymorphisms of the OPRM1, DRD2, COMT, ABCB1, CYP2B6, CYP3A5, CYP2C19 and CYP2D6 genes. RESULTS: Methadone maintenance dose was correlated to the highest dose ever used (r(2) = 0.57, P < 0.0001). Fractioned methadone intake (odds ratio 4.87, 95% confidence interval 1.27-18.6, P = 0.02), bodyweight (odds ratio 1.57, 95% confidence interval 1.01-2.44, P = 0.04), history of cocaine dependence (80 vs. 44 mg day(-1) in never-addict patients, P = 0.005) and ethnicity (Asian > Caucasian > African, P = 0.04) were independently associated with high-dose methadone in multiple regression analysis. A modest correlation was observed between liver/intestinal CYP3A4 activity and methadone dose at steady state (Spearman rank correlation coefficient [rs ] = 0.21, P = 0.06) but not with highest dose ever used (rs = 0.15, P = 0.18) or dose-normalized R,S-methadone trough concentrations (rs = -0.05, P = 0.64). Concomitant CYP3A4 inhibitors only affected the relationship between methadone dose and R,S-methadone trough concentration. None of the genetic polymorphisms explored was predictive of the methadone maintenance dose. CONCLUSIONS: Methadone maintenance dose was predicted by sociodemographic and clinical variables rather than genetic polymorphisms or liver/intestinal CYP3A4 activity in stable patients.

Carrier-mediated cocaine transport at the blood-brain barrier as a putative mechanism in addiction liability.

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.

A physiologically based modeling strategy during preclinical CNS drug development.

Physiologically based pharmacokinetic (PBPK) modeling of the central nervous system (CNS) provides the opportunity to predict the relevant drug concentrations at the therapeutic target site during preclinical and clinical development. In order to successfully interpret model results, and to provide confidence in the subsequent human predictions, it is essential that an appropriate model structure is chosen at the preclinical stage which takes into account both physiological and drug-specific knowledge. However, the models published to date in the literature show significant variation in the approaches applied by different authors, which can lead to difficulties in the interpretation of model parameter estimates. We aimed to develop a coherent PBPK modeling approach in the rat, which would also be adaptable depending on the quantity and quality of in vivo data obtained during drug development. Based on a sensitivity analysis of the model parameters, and using three CNS drugs as case studies (atomoxetine, acetaminophen, and S 18986), we proposed a decision tree to aid in the appropriate parametrization and structure of the model according to the data available. We compared our parameter estimates to those originally published, and considered the impact of the respective approaches on the mechanistic interpretation of the parameter values. Since the measurement of brain extracellular fluid (ECF) concentrations using microdialysis is not routinely performed in the industrial environment, we also evaluated the bottom-up scaling of in vitro permeability data from the Caco-2 cell line to predict BBB passive permeability in the absence of measured ECF concentrations. Our strategy demonstrates the value of PBPK as a prediction tool throughout the development process of CNS-targeting drugs.

Comparing translational population-PBPK modelling of brain microdialysis with bottom-up prediction of brain-to-plasma distribution in rat and human.

The prediction of brain extracellular fluid (ECF) concentrations in human is a potentially valuable asset during drug development as it can provide the pharmacokinetic input for pharmacokinetic-pharmacodynamic models. This study aimed to compare two translational modelling approaches that can be applied at the preclinical stage of development in order to simulate human brain ECF concentrations. A population-PBPK model of the central nervous system was developed based on brain microdialysis data, and the model parameters were translated to their corresponding human values to simulate ECF and brain tissue concentration profiles. In parallel, the PBPK modelling software Simcyp was used to simulate human brain tissue concentrations, via the bottom-up prediction of brain tissue distribution using two different sets of mechanistic tissue composition-based equations. The population-PBPK and bottom-up approaches gave similar predictions of total brain concentrations in both rat and human, while only the population-PBPK model was capable of accurately simulating the rat ECF concentrations. The choice of PBPK model must therefore depend on the purpose of the modelling exercise, the in vitro and in vivo data available and knowledge of the mechanisms governing the membrane permeability and distribution of the drug.

Quantitative targeted absolute proteomic analysis of transporters, receptors and junction proteins for validation of human cerebral microvascular endothelial cell line hCMEC/D3 as a human blood-brain barrier model.

Human cerebral microvascular endothelial cell line hCMEC/D3 is an established model of the human blood-brain barrier (BBB). The purpose of the present study was to determine, by means of quantitative targeted absolute proteomics, the protein expression levels in hCMEC/D3 cells of multiple transporters, receptors and junction proteins for comparison with our previously reported findings in isolated human brain microvessels. Among 91 target molecules, 12 transporters, 2 receptors, 1 junction protein and 1 membrane marker were present at quantifiable levels in plasma membrane fraction of hCMEC/D3 cells. ABCA2, MDR1, MRP4, BCRP, GLUT1, 4F2hc, MCT1, ENT1, transferrin and insulin receptors and claudin-5 were detected in both hCMEC/D3 cells and human brain microvessels. After normalization based on Na(+)/K(+) ATPase expression, the differences in protein expression levels between hCMEC/D3 cells and human brain microvessels were within 4-fold for these proteins, with the exceptions of ENT1, transferrin receptor and claudin-5. ABCA8, LAT1, LRP1 and γ-GTP were below the limit of quantification in the cells, but were found in human brain microvessels. ABCA3, ABCA6, MRP1 and ATA1 were found only in hCMEC/D3 cells. Furthermore, compared with human umbilical vein endothelial cells (HUVECs) as reference nonbrain endothelial cells, MDR1 was found only in hCMEC/D3 cells, and GLUT1 expression was 15-fold higher in hCMEC/D3 cells than in HUVECs. In conclusion, this is the first study to examine the suitability and limitations of the hCMEC/D3 cell line as a BBB functional model in terms of quantitative expression levels of transporters, receptors and tight junction proteins.

Methodologies to assess drug permeation through the blood-brain barrier for pharmaceutical research.

The drug discovery process for drugs that target the central nervous system suffers from a very high rate of failure due to the presence of the blood-brain barrier, which limits the entry of xenobiotics into the brain. To minimise drug failure at different stages of the drug development process, new methodologies have been developed to understand the absorption, distribution, metabolism, excretion and toxicity (ADMET) profile of drug candidates at early stages of drug development. Additionally, understanding the permeation of drug candidates is also important, particularly for drugs that target the central nervous system. During the first stages of the drug discovery process, in vitro methods that allow for the determination of permeability using high-throughput screening methods are advantageous. For example, performing the parallel artificial membrane permeability assay followed by cell-based models with interesting hits is a useful technique for identifying potential drugs. In silico models also provide interesting information but must be confirmed by in vitro models. Finally, in vivo models, such as in situ brain perfusion, should be studied to reduce a large number of drug candidates to a few lead compounds. This article reviews the different methodologies used in the drug discovery and drug development processes to determine the permeation of drug candidates through the blood-brain barrier.

Induction of P-glycoprotein and Bcrp at the rat blood-brain barrier following a subchronic morphine treatment is mediated through NMDA/COX-2 activation.

Subchronic morphine treatment induces P-glycoprotein (P-gp) up-regulation at the blood-brain barrier. This study investigates the rate and extent to which P-gp and breast cancer-resistance protein (Bcrp) increase at the rat blood-brain barrier following subchronic morphine treatment. Rats were given increasing doses of morphine (10-40 mg/kg) or saline i.p. twice daily for 5 days. The brain cortex large vessels and microvessels were then mechanical isolated 6, 9, 12, 24, and 36 h after the last injection. The gene and protein expression of P-gp and Bcrp in morphine-treated and control rats were compared by qRT-PCR and western blotting. The levels of Mdr1a and Bcrp mRNAs were not significantly modified 6 h post morphine, but the Mdr1a mRNA increased 1.4-fold and Bcrp mRNA 2.4-fold at 24 h. P-gp and Bcrp protein expression in brain microvessels was unchanged 6 h post morphine and increased 1.5-fold at 24 h. This effect was more pronounced in large vessels than in microvessels. However, extracellular morphine concentrations of 0.01-10 µM did not modify the expressions of the MDR1 and BCRP genes in hCMEC/D3 human endothelial brain cells in vitro. MK-801 (NMDA antagonist) and meloxicam (cyclo-oxygenase-2 inhibitor) given after morphine treatment completely blocked P-gp and Bcrp up-regulation. Interestingly, misoprostol and iloprost, two well-known agonists of prostaglandin E2 receptors induced both MDR1 and BCRP mRNA levels in hCMEC/D3. Thus, morphine does not directly stimulate P-gp and Bcrp expression by the brain endothelium, but glutamate released during morphine withdrawal may do so by activating the NMDA/cyclo-oxygenase-2 cascade.

Respiratory toxicity of buprenorphine results from the blockage of P-glycoprotein-mediated efflux of norbuprenorphine at the blood-brain barrier in mice.

OBJECTIVES: Deaths due to asphyxia as well as following acute poisoning with severe respiratory depression have been attributed to buprenorphine in opioid abusers. However, in human and animal studies, buprenorphine exhibited ceiling respiratory effects, whereas its metabolite, norbuprenorphine, was assessed as being a potent respiratory depressor in rodents. Recently, norbuprenorphine, in contrast to buprenorphine, was shown in vitro to be a substrate of human P-glycoprotein, a drug-transporter involved in all steps of pharmacokinetics including transport at the blood-brain barrier. Our objectives were to assess P-glycoprotein involvement in norbuprenorphine transport in vivo and study its role in the modulation of buprenorphine-related respiratory effects in mice. SETTING: University-affiliated research laboratory, INSERM U705, Paris, France. SUBJECTS: Wild-type and P-glycoprotein knockout female Friend virus B-type mice. INTERVENTIONS: Respiratory effects were studied using plethysmography and the P-glycoprotein role at the blood-brain barrier using in situ brain perfusion. MEASUREMENTS AND MAIN RESULTS: Norbuprenorphine(≥ 1 mg/kg) and to a lesser extent buprenorphine (≥ 10 mg/kg) were responsible for dose-dependent respiratory depression combining increased inspiratory (TI) and expiratory times (TE). PSC833, a powerful P-glycoprotein inhibitor, significantly enhanced buprenorphine-related effects on TI (p < .01) and TE (p < .05) and norbuprenorphine-related effects on minute volume (VE, p < .05), TI, and TE (p < .001). In P-glycoprotein-knockout mice, buprenorphine-related effects on VE (p < .01), TE (p < .001), and TI (p < .05) and norbuprenorphine-related effects on VE (p < .05) and TI (p < .001) were significantly enhanced. Plasma norbuprenorphine concentrations were significantly increased in PSC833-treated mice (p < .001), supporting a P-glycoprotein role in norbuprenorphine pharmacokinetics. Brain norbuprenorphine efflux was significantly reduced in PSC833-treated and P-glycoprotein-knockout mice (p < .001), supporting P-glycoprotein-mediated norbuprenorphine transport at the blood-brain barrier. CONCLUSIONS: P-glycoprotein plays a key-protective role in buprenorphine-related respiratory effects, by allowing norbuprenorphine efflux at the blood-brain barrier. Our findings suggest a major role for drug-drug interactions that lead to P-glycoprotein inhibition in buprenorphine-associated fatalities and respiratory depression.

Discrepancies in the P-glycoprotein-mediated transport of (18)F-MPPF: a pharmacokinetic study in mice and non-human primates.

PURPOSE: Several in vivo studies have found that the 5-HT(1A) PET radioligand (18)F-MPPF is a substrate of rodent P-glycoprotein (P-gp). However, in vitro assays suggest that MPPF is not a substrate of human P-gp. We have now tested the influence of inhibiting P-gp on the brain kinetics of (18)F-MPPF in mice and non-human primates. METHODS: We measured the peripheral kinetics (arterial input function, metabolism, free fraction in plasma (f(P))) during (18)F-MPPF brain PET scanning in baboons with or without cyclosporine A (CsA) infusion. We measured (3)H-MPPF transport at the mouse BBB using in situ brain perfusion in P-gp/Bcrp deficient mice and after inhibiting P-gp with PSC833. RESULTS: There was an unexpected 1.9-fold increase in brain area under the curve in CsA-treated baboons (n = 4), with no change in radiometabolite-corrected arterial input. However, total volume of distribution corrected for f(P) (V(T)/f(P)) remained unchanged. In situ brain perfusion showed that P-gp restricted the permeability of the mouse BBB to (3)H-MPPF while Bcrp did not. CONCLUSION: These and previous in vitro results suggest that P-gp may not influence the permeability of human BBB to (18)F-MPPF. However, CsA treatment increased (18)F-MPPF free fraction, which is responsible for a misleading, P-gp unrelated enhanced brain uptake.

Transcriptomic and quantitative proteomic analysis of transporters and drug metabolizing enzymes in freshly isolated human brain microvessels.

We have investigated the transcriptomic and/or proteomic patterns of 71 solute carrier (SLC) and organic solute (OST) transporters, 34 ATP-binding cassette (ABC) transporters, and 51 metabolizing enzymes in human brain microvessels. We used quantitative RT-PCR and LC-MS/MS to examine isolated brain microvessels and cortex biopsies from 12 patients with epilepsia or glioma. SLC2A1/GLUT1, SLC1A3/EAAT1, and SLC1A2/EAAT2 were the main SLC proteins whereas ABCG2/BCRP, ABCB1/MDR1, ABCA2 and ABCA8 were the main ABC quantified in isolated brain microvessels; ABCG2/BCRP was 1.6-fold more expressed than ABCB1/MDR1, and ABCC4/MRP4 was 10 times less abundant than ABCB1/MDR1. CYP1B1 and CYP2U1 were the only quantifiable CYPs. Finally, GSTP1, COMT, GSTM3, GSTO1 and GSTM2 proteins were the main phase II enzymes quantified; UGTs and NATs were not detected. Our extensive investigation of gene and protein patterns of transporters and metabolizing enzymes provides new molecular information for understanding drug entry and metabolism in the human blood-brain barrier.