Successful Prediction of Human Steady-State Unbound Brain-to-Plasma Concentration Ratio of P-gp Substrates Using the Proteomics-Informed Relative Expression Factor Approach.

In order to optimize central nervous system (CNS) drug development, accurate prediction of the drug's human steady-state unbound brain interstitial fluid-to-plasma concentration ratio (Kp,uu,brain ) is critical, especially for drugs that are effluxed by the multiple drug resistance transporters (e.g., P-glycoprotein, P-gp). Due to lack of good in vitro human blood-brain barrier models, we and others have advocated the use of a proteomics-informed relative expressive factor (REF) approach to predict Kp,uu,brain . Therefore, we tested the success of this approach in humans, with a focus on P-gp substrates, using brain positron emission tomography imaging data for verification. To do so, the efflux ratio (ER) of verapamil, N-desmethyl loperamide, and metoclopramide was determined in human P-gp-transfected MDCKII cells using the Transwell assay. Then, using the ER estimate, Kp,uu,brain of the drug was predicted using REF (ER approach). Alternatively, in vitro passive and P-gp-mediated intrinsic clearances (CLs) of these drugs, estimated using a five-compartmental model, were extrapolated to in vivo using REF (active CL) and brain microvascular endothelial cells protein content (passive CL). The ER approach successfully predicted Kp,uu,brain of all three drugs within twofold of observed data and within 95% confidence interval of the observed data for verapamil and N-desmethyl loperamide. Using the in vitro-to-in vivo extrapolated clearance approach, Kp,uu,brain was reasonably well predicted but not the brain unbound interstitial fluid drug concentration-time profile. Therefore, we propose that the ER approach be used to predict Kp,uu,brain of CNS candidate drugs to enhance their success in development.

Role of P-gp and HDAC2 and their Reciprocal Relationship in Uncontrolled Asthma.

INTRODUCTION: Resistance to corticosteroid is an essential mechanism in uncontrolled asthma as the corticosteroid is the mainstay of therapy. There are recent reports that epigenetic factors play a crucial role in the regulation of steroid action. Overexpression of P glycoprotein (P-gp) and reduced expression of Histone Deacetylase 2 (HDAC2) have been linked to regulating the steroid action in other diseases like Nephrotic Syndrome (NS). However, their role in uncontrolled asthma is still not clear and warrants further investigation. We evaluated the expression and activity of P-gp and HDAC2 in patients with Controlled Asthma (CA) and Uncontrolled Asthma (UA). METHODS: A total of 60 CA (mean age 51.72±17.02 years, male=38), and 38 of UA (mean age=53.55±11.90 years, male=17) were recruited. The level of control was defined according to (Global Initiative for Asthma) GINA 2016 criteria. The mRNA expression of HDAC2 and P-gp was studied by quantitative real-time Polymerase Chain Reaction (PCR), the functional activity of P-gp was evaluated by a commercially available kit via flow cytometry, and HDAC2 enzymatic activity was measured by commercially available kit by Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: P-gp expression and the functionality were significantly higher in the UA group of patients as compared to the CA group of patients (p<0.005), moreover HDAC2 expression was significantly reduced in UA patients as compared to CA patients, (p<0.005). The enzymatic activity of HDAC2 was also significantly reduced in UA patients as compared to CA patients (p<0.005). CONCLUSION: P-gp overexpression and HDAC2 under expression play an essential role in uncontrolled asthma by impairing the response to corticosteroid.

Prevalence of p-glycoprotein (PGP) expression, function and its effect on efficacy of rifampicin in patients with lymph node tuberculosis.

OBJECTIVE: P-glycoprotein (PGP) overexpression may be one of the operating mechanisms of suboptimal responses to antitubercular treatment (ATT) in patients with lymph node tuberculosis. This might become responsible for the development of drug resistance later due to exposure of subtherapeutic concentrations to the mycobacteria. In this study we aim to study the prevalence of PGP expression and function and its relationship with serum concentrations of Rifampicin in consecutive patients with lymph node tuberculosis. METHODS: All newly diagnosed treatment naïve subjects with a confirmed diagnosis of tubercular lymphadenopathy were included in the study and the expression and function of PGP in blood was determined by flowcytometry at baseline and after two months of treatment. Serum levels of Rifampicin was measured at 2 months by high performance liquid chromatography (HPLC). The mean net PGP expression expressed as percent and relative fluorescence indices (RFI) of PGP expression and function respectively was compared at baseline at 2 months and was also correlated with serum rifampicin levels. RESULTS: The mean net PGP expression, RFI of PGP expression and RFI of PGP function were significantly higher in patients with lymph node tuberculosis as compared to healthy controls and the mean net PGP expression and RFI of PGP expression were significantly higher at 2 months as compared to baseline (25.64 ± 5.18% vs. 27.68 ± 4.89%, 4.34 ± 1.09% vs. 4.95 ± 1.55). There was no significant difference in RFI of PGP expression and RFI of PGP function between the poor-responders and responders at baseline and 2 months however there was a trend towards significantly higher net PGP expression amongst poor responders at baseline. The mean serum rifampicin levels were 10.74 ± 2.36 µg/ml in the responder group and 7.86 ± 1.21 µg/ml in the non-responder group and the difference between the two was statistically significant (p = 0.004). CONCLUSIONS: Overexpression of PGP is common in patients with lymph node tuberculosis and leads to lower concentrations of Rifampicin in blood which subsequently may give rise to development of drug resistance. This is also responsible for poor therapeutic responses in these patients. Nonspecific inhibitors of PGP may be used in conjunction with ATT to augment therapeutic response in such cases.

Correlation of levetiracetam concentration in peripheral blood mononuclear cells with clinical efficacy: A sensitive monitoring biomarker in patients with epilepsy.

PURPOSE: Therapeutic drug monitoring (TDM) is increasingly recommended in antiepileptic drug (AED) therapy, yet a complex relationship exists between the unbound-drug serum concentration (Cu.serum) as a monitoring biomarker and clinical efficacy. The study was designed to investigate the validity of the intracellular unbound-drug concentration in Peripheral Blood Mononuclear Cells (Cu.PBMC) as a feasible TDM tool in relation to levetiracetam (LEV). METHODS: Patients from epilepsy out-patient centre were included in a 4-month descriptive prospective study. Trough serum and PBMC LEV concentrations were monthly determined using HPLC and correlated with clinical features, demographic data, and P-glycoprotein (P-gp) expression. RESULTS: Seventy-patients completed the study with a LEV dose range of 500-3000 mg/day. An absolute range for LEV Cu.serum and Cu.PBMC was 1.00-26.99 and 0.33-4.43 µg/mL, respectively. Unlike Cu.serum, the average four-month LEV Cu.PBMC displayed a significant positive correlation with clinical features and P-gp expression; where patients with higher LEV Cu.PBMC experienced less number of seizure/month, better cognition and quality of life, and had a more reduction in P-gp expression, but no significant correlation with LEV daily dose was observed. A therapeutic response threshold of ≥ 0.82 µg/mL for LEV Cu.PBMCwas perceived by using a receiver operating characteristic curve that related the number of seizure/month to the LEV Cu.PBMC. The validity of this therapeutic threshold was significant in contrast to LEV Cu.serum. CONCLUSION: Levetiracetam PBMC concentration is a more sensitive biomarker for LEV efficacy and correlates better with clinical events than Cu.serum and could represent a novel tool for more precise LEV monitoring.

Phenotypic Assessment of Drug Metabolic Pathways and P-Glycoprotein in Patients Treated With Antidepressants in an Ambulatory Setting.

OBJECTIVE: Drug-metabolizing enzymes (DMEs), such as cytochrome P450 (CYP) enzymes, and transporters have emerged as major determinants of variability in drug metabolism and response. This study investigated the association between CYP and P-glycoprotein activities and plasma antidepressant concentration in an outpatient clinical setting. Secondary outcomes were antidepressant efficacy and tolerance. We also describe phenotypes in patients treated with antidepressants and evaluate the tolerance of a minimally invasive phenotyping approach. METHODS: From January 2015 to August 2015, 64 patients on a stable antidepressant regimen underwent a simultaneous assessment of steady-state antidepressant concentration and DME (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A) and P-glycoprotein transporter activity using a cocktail phenotyping approach. Psychiatric diagnoses were in accordance with DSM-5. RESULTS: We observed a high proportion of subjects (> 20%) with reduced activity of CYP2C19, CYP2D6, CYP3A4, and P-glycoprotein. As expected, higher CYP activity for major metabolic pathways was associated with lower concentration of the parent compound (CYP2C19 and escitalopram, P = .025; CYP2D6 and fluoxetine, P < .001; CYP2C19 and sertraline, P = .001), higher concentration of the metabolite (CYP2D6 and O-desmethylvenlafaxine, P = .007), and higher metabolite-to-parent drug ratio (CYP2C19 and escitalopram, P = .03; CYP2D6 and fluoxetine, P < .001; CYP2C19 and sertraline, P = .048; CYP2B6 and sertraline, P = .006). Phenotyping also highlighted the relevance of a minor metabolic pathway for venlafaxine (CYP3A4). Insufficient response and adverse reactions to antidepressants were not significantly associated with plasma antidepressant concentration, DME, or P-glycoprotein activity. Tolerance of the phenotypic test in ambulatory settings was found to be excellent. CONCLUSIONS: The phenotypic assessment of DMEs and a transporter is a valuable, well-tolerated method to explore the interindividual variability in drug disposition in clinical settings. The method is able to account for the inhibitory activity of antidepressants themselves and for polymedication, which is frequent in this population of refractory depressed patients. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02438072.

Serum levels of P-glycoprotein and persistence of disease activity despite treatment in patients with systemic lupus erythematosus.

Around 25% of patients with systemic lupus erythematosus (SLE) could be refractory to conventional therapies. P-glycoprotein expression on cell surface has been implied on drug resistance, however, to date, it is unknown if P-gp serum levels are associated with SLE disease activity. Evaluate the association of serum P-gp levels and SLE with disease activity despite treatment. A cross-sectional study was conducted on 93 female SLE patients, all receiving glucocorticoids at stable doses for the previous 6 months before to baseline. SLE patients were classified into two groups: (a) patients with active disease [SLE disease activity index (SLEDAI) ≥ 3] despite treatment, and (b) patients with inactive disease (SLEDAI < 3) after treatment. Forty-three healthy females comprised the control group. Serum P-gp, anti-DNA, and both anti-nucleosome antibody levels were measured using ELISA. Active-SLE patients despite treatment had higher P-gp levels compared with inactive-SLE after treatment (78.02 ng/mL ± 114.11 vs. 33.75 ng/mL ± 41.11; p = 0.018) or versus reference group subjects (30.56 ng/mL ± 28.92; p = 0.011). P-gp levels correlated with the scores of SLEDAI (r = 0.26; p = 0.01), Mexican-SLEDAI (MEX-SLEDAI) (r = 0.32; p = 0.002), SLICC/ACR damage index (r = 0.47; p < 0.001), and with prednisone doses (r = 0.33; p = 0.001). In the multivariate model, the high P-gp levels were associated with SLICC/ACR score (p = 0.001), and SLEDAI score (p = 0.014). Our findings support a relationship between serum P-gp levels and SLE with disease activity despite treatment, but it requires further validation in longitudinal studies.

Quantification of Transporter and Receptor Proteins in Dog Brain Capillaries and Choroid Plexus: Relevance for the Distribution in Brain and CSF of Selected BCRP and P-gp Substrates.

Transporters at the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) play a pivotal role as gatekeepers for efflux or uptake of endogenous and exogenous molecules. The protein expression of a number of them has already been determined in the brains of rodents, nonhuman primates, and humans using quantitative targeted absolute proteomics (QTAP). The dog is an important animal model for drug discovery and development, especially for safety evaluations. The purpose of the present study was to clarify the relevance of the transporter protein expression for drug distribution in the dog brain and CSF. We used QTAP to examine the protein expression of 17 selected transporters and receptors at the dog BBB and BCSFB. For the first time, we directly linked the expression of two efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), to regional brain and CSF distribution using specific substrates. Two cocktails, each containing one P-gp substrate (quinidine or apafant) and one BCRP substrate (dantrolene or daidzein) were infused intravenously prior to collection of the brain. Transporter expression varied only slightly between the capillaries of different brain regions and did not result in region-specific distribution of the investigated substrates. There were, however, distinct differences between brain capillaries and choroid plexus. Largest differences were observed for BCRP and P-gp: both were highly expressed in brain capillaries, but no BCRP and only low amounts of P-gp were detected in the choroid plexus. Kp,uu,brain and Kp,uu,CSF of both P-gp substrates were indicative of drug efflux. Also, Kp,uu,brain for the BCRP substrates was low. In contrast, Kp,uu,CSF for both BCRP substrates was close to unity, resulting in Kp,uu,CSF/Kp,uu,brain ratios of 7 and 8, respectively. We conclude that the drug transporter expression profiles differ between the BBB and BCSFB in dogs, that there are species differences in the expression profiles, and that CSF is not a suitable surrogate for unbound brain concentrations of BCRP substrates in dogs.

Role of P-glycoprotein inhibitors in children with drug-resistant epilepsy.

OBJECTIVE: The role of P-glycoprotein (Pgp), one of the known multidrug transporters, has been suggested in drug-resistant epilepsy (DRE). The following study aimed to measure the serum level of Pgp as a possible indicator of tissue Pgp overexpression in patients with DRE and to assess the efficacy of verapamil (as a Pgp inhibitor agent) in these patients. MATERIAL AND METHODS: A group of 24 patients with DRE were recruited and subdivided into two groups, one receiving verapamil and the other receiving a placebo in a double-blind randomized study. Pgp serum levels were measured at enrollment and 12 months later. Twenty medically controlled epileptic patients served as a control group. RESULTS: A significant statistical increase was found in the Pgp level of patients when compared the control group. Patients on both verapamil and the placebo showed improvement in seizure frequency and severity where statistical analysis showed no significant differences. CONCLUSION: Pgp serum levels in patients with DRE were significantly elevated compared to patients with medically controlled epilepsy. The effect of verapamil as Pgp inhibitor on DRE requires further evaluation and research.

Clinical relevance of P-glycoprotein activity on peripheral blood mononuclear cells and polymorphonuclear neutrophils to methotrexate in systemic lupus erythematosus patients.

To elucidate the relationship between P-glycoprotein activity on peripheral blood leukocytes of systemic lupus erythematosus (SLE) patients with lupus arthritis and the clinical response to methotrexate. An observational study was made in patients with SLE according to ACR criteria 1997 who had arthralgia and arthritis and received methotrexate for ≥3 months. Methotrexate responders and non-responders were compared according to the Clinical Disease Activity Index. Mononuclear cells and polymorphonuclear neutrophils were isolated from SLE patients and P-glycoprotein expression was measured using the relative fluorescence index and percentage of positive cells. The chi-square test was used to compare P-glycoprotein activity between responders and non-responders. Thirty-two patients with a mean age of 45.4 ± 10.7 years were included: 34.4% had a response to methotrexate and 65.6% did not. Mean relative fluorescence units of both mononuclear cells and polymorphonuclear neutrophils were significantly lower in patients with a good response (7.0 ± 4.3 vs. 9.6 ± 3.8; p = 0.041 and 4.2 ± 3.5 vs. 7.6 ± 4.0; p = 0.004). The prevalence of low fluorescence levels (<6 relative fluorescence units), signifying higher P-glycoprotein activity of both mononuclear cells and polymorphonuclear neutrophils, was higher in methotrexate responders than in non-responders (27.3 vs. 4.8%; p = 0.10 and 81.8 vs. 23.8%; p = 0.003, respectively). In SLE patients with joint involvement treated with methotrexate, P-glycoprotein activity was higher in responders to methotrexate than in non-responders. Further studies are required to determine the mechanisms behind this finding and whether P-glycoprotein activity mediates alterations in methotrexate efficacy.

Coadministration of Rifampin Significantly Reduces Odanacatib Concentrations in Healthy Subjects.

This open-label 2-period study assessed the effect of multiple-dose administration of rifampin, a strong cytochrome P450 3A (CYP3A) and P-glycoprotein inducer, on the pharmacokinetics of odanacatib, a cathepsin K inhibitor. In period 1, 12 healthy male subjects (mean age, 30 years) received a single dose of odanacatib 50 mg on day 1, followed by a 28-day washout. In period 2, subjects received rifampin 600 mg/day for 28 days; odanacatib 50 mg was coadministered on day 14. Blood samples for odanacatib pharmacokinetics were collected at predose and on day 1 of period 1 and day 14 of period 2. Coadministration of odanacatib and rifampin significantly reduced odanacatib exposure. The odanacatib AUC0-∞ geometric mean ratio (90% confidence interval) of odanacatib + rifampin/odanacatib alone was 0.13 (0.11-0.16). The harmonic mean ± jackknife standard deviation apparent terminal half-life (t½ ) was 71.6 ± 10.2 hours for odanacatib alone and 16.0 ± 3.4 hours for odanacatib + rifampin, indicating greater odanacatib clearance following coadministration with rifampin. Samples were collected in period 2 during rifampin dosing (days 1, 14, and 28) and after rifampin discontinuation (days 35, 42, and 56) to evaluate the ratio of plasma 4ß-hydroxycholesterol to total serum cholesterol as a CYP3A4 induction biomarker; the ratio increased ∼5-fold over 28 days of daily dosing with 600 mg rifampin, demonstrating sensitivity to CYP3A4 induction.

Assessment of P-Glycoprotein Transport Activity at the Human Blood-Retina Barrier with (R)-11C-Verapamil PET.

P-glycoprotein (ABCB1) is expressed at the blood-retina barrier (BRB), where it may control distribution of drugs from blood to the retina and thereby influence drug efficacy and toxicity. Methods: We performed PET scans with the ABCB1 substrate (R)-11C-verapamil on 5 healthy male volunteers without and with concurrent infusion of the ABCB1 inhibitor tariquidar. We estimated the rate constants for radiotracer transfer across the BRB (K1, k2) and total retinal distribution volume VTResults: During ABCB1 inhibition, retinal VT and influx rate constant K1 were significantly, by 1.4 ± 0.5-fold and 1.5 ± 0.3-fold, increased compared with baseline. Retinal efflux rate constant k2 was significantly decreased by 2.8 ± 1.0-fold. Conclusion: We found a significant increase in (R)-11C-verapamil distribution to the retina during ABCB1 inhibition, which provides first in vivo evidence for ABCB1 transport activity at the human BRB. The increase in retinal distribution was approximately 2.5-fold less pronounced than previously reported for the blood-brain barrier.

QSAR Model of Unbound Brain-to-Plasma Partition Coefficient, Kp,uu,brain: Incorporating P-glycoprotein Efflux as a Variable.

We report development and prospective validation of a QSAR model of the unbound brain-to-plasma partition coefficient, Kp,uu,brain, based on the in-house data set of ∼1000 compounds. We discuss effects of experimental variability, explore the applicability of both regression and classification approaches, and evaluate a novel, model-within-a-model approach of including P-glycoprotein efflux prediction as an additional variable. When tested on an independent test set of 91 internal compounds, incorporation of P-glycoprotein efflux information significantly improves the model performance resulting in an R2 of 0.53, RMSE of 0.57, Spearman's Rho correlation coefficient of 0.73, and qualitative prediction accuracy of 0.8 (kappa = 0.6). In addition to improving the performance, one of the key advantages of this approach is the larger chemical space coverage provided indirectly through incorporation of the in vitro, higher throughput data set that is 4 times larger than the in vivo data set.

Association of ACE and MDR1 Gene Polymorphisms with Steroid Resistance in Children with Idiopathic Nephrotic Syndrome.

AIM: The purpose of the study was to investigate the distribution of insertion/deletion (I/D) polymorphisms of the angiotensin-converting enzyme (ACE) gene and three exonic polymorphisms of the multidrug resistance 1 (MDR1) gene (C3435T, C1236T, and G2677T) in children diagnosed with idiopathic nephrotic syndrome (INS). MATERIALS AND METHODS: The study group consisted of 100 healthy controls and 150 INS patients, of which 50 were steroid resistant. Genomic DNA from blood samples was isolated from both of these groups and genotyping of the ACE and MDR1 genes was performed by polymerase chain reaction (PCR) using specific primers. RESULTS: There was no significant difference observed in the genotypic distribution and D allele frequency of the ACE gene. The two single-nucleotide polymorphisms (SNPs), C1236T and C3435T, of the MDR1 gene showed no significance, whereas the SNP G2677T/A was significantly associated with the genotypes GT and GA of the MDR1 gene, indicating it may be a potential marker to detect drug resistance. CONCLUSION: Screening these polymorphisms will pave the way to better understand the molecular mechanisms of the disease, which may be useful in developing targeted therapies for INS patients.

Serum exosomal P-glycoprotein is a potential marker to diagnose docetaxel resistance and select a taxoid for patients with prostate cancer.

OBJECTIVES: Docetaxel is used as the first-line chemotherapy for castration-resistant prostate cancer (CRPC), but docetaxel resistance occurs in part owing to induction of P-glycoprotein (P-gp) encoded by multidrug resistance protein 1 (MDR1) gene. A recently developed taxane-cabazitaxel-has poor affinity for P-gp and is thereby effective in docetaxel-resistant CRPC. It has been recently demonstrated that exosomes in the body fluids could serve as a diagnostic marker because they contain proteins and RNAs specific to the cells from which they are derived. In this study, we aimed to investigate if P-gp in blood exosomes could be a marker to diagnose docetaxel resistance and select a taxoid for patients with CRPC. METHODS AND MATERIALS: Exosomes were isolated by differential centrifugation from docetaxel-resistant prostate cancer (PC-3) cells (PC-3R) and their parental PC-3 cells and from the serum of patients. Silencing of P-gp was performed by small interfering RNA transfection. Protein expression was examined by Western blot analysis. Viability of cells treated with docetaxel or cabazitaxel was determined by water soluble tetrazolium salt (WST) assay. RESULTS: The level of P-gp was higher in exosomes as well as cell lysates from PC-3R cells than in those from PC-3 cells. Cabazitaxel effectively killed PC-3R cells, and MDR1 knockdown improved the sensitivity of PC-3R cells to docetaxel but not to cabazitaxel. The P-gp level in blood exosomes was relatively higher in clinically docetaxel-resistant patients than in therapy-naïve patients. CONCLUSIONS: Our results suggest that detection of P-gp in blood exosomes, which is involved in resistance to docetaxel but not to cabazitaxel, could be useful to diagnose docetaxel resistance and select an appropriate taxoid for patients with CRPC-docetaxel or cabazitaxel.

A robust and versatile signal-on fluorescence sensing strategy based on SYBR Green I dye and graphene oxide.

A robust and versatile signal-on fluorescence sensing strategy was developed to provide label-free detection of various target analytes. The strategy used SYBR Green I dye and graphene oxide as signal reporter and signal-to-background ratio enhancer, respectively. Multidrug resistance protein 1 (MDR1) gene and mercury ion (Hg(2+)) were selected as target analytes to investigate the generality of the method. The linear relationship and specificity of the detections showed that the sensitive and selective analyses of target analytes could be achieved by the proposed strategy with low detection limits of 0.5 and 2.2 nM for MDR1 gene and Hg(2+), respectively. Moreover, the strategy was used to detect real samples. Analytical results of MDR1 gene in the serum indicated that the developed method is a promising alternative approach for real applications in complex systems. Furthermore, the recovery of the proposed method for Hg(2+) detection was acceptable. Thus, the developed label-free signal-on fluorescence sensing strategy exhibited excellent universality, sensitivity, and handling convenience.

Geneva cocktail for cytochrome p450 and P-glycoprotein activity assessment using dried blood spots.

The suitability of the capillary dried blood spot (DBS) sampling method was assessed for simultaneous phenotyping of cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp) using a cocktail approach. Ten volunteers received an oral cocktail capsule containing low doses of the probes bupropion (CYP2B6), flurbiprofen (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A), and fexofenadine (P-gp) with coffee/Coke (CYP1A2) on four occasions. They received the cocktail alone (session 1), and with the CYP inhibitors fluvoxamine and voriconazole (session 2) and quinidine (session 3). In session 4, subjects received the cocktail after a 7-day pretreatment with the inducer rifampicin. The concentrations of probes/metabolites were determined in DBS and plasma using a single liquid chromatography-tandem mass spectrometry method. The pharmacokinetic profiles of the drugs were comparable in DBS and plasma. Important modulation of CYP and P-gp activities was observed in the presence of inhibitors and the inducer. Minimally invasive one- and three-point (at 2, 3, and 6 h) DBS-sampling methods were found to reliably reflect CYP and P-gp activities at each session.

The impact of genetic polymorphisms on time required to attain the target tacrolimus levels and subsequent pharmacodynamic outcomes in pediatric kidney transplant patients.

Recent pharmacogenetic studies involving various transplant recipients in diverse ethnic populations revealed contradictory findings regarding the impact of CYP3A5 and multidrug resistance-1 (MDR1) single nucleotide polymorphisms (SNPs) on the pharmacodynamics of tacrolmius (TAC). The aim of the current study was to evaluate the impact of these SNPs on the time required to attain target TAC levels and subsequent pharmacodynamic outcomes in pediatric kidney transplant patients. Thirty-eight patients were genotyped for CYP3A5FNx011 and FNx013, and MDR1 C3435T. Notably, none of the patients expressing CYP3A5 FNx011FNx011 or FNx011FNx013 achieved the target concentration of 10-20 ng/mL within the first 2 weeks or even the first month after transplant. However, 34.4% of CYP3A5 FNx013FNx013 achieved and maintained the target goal within the first and second weeks (P <0.05). In contrast, C3435T polymorphism had no significant influence on the proportion of patients achieving target TAC levels. An examination of the impact of genotype combinations on clinical outcomes revealed an increased incidence of acute, chronic rejection and graft loss in patients carrying heterozygous MDR1 C3435T alleles (CT); this indicates the possibility of an additive effect of this SNP on its concurrent combination with a FNx013FNx013 SNP. In conclusion, our study revealed trends toward unwanted outcomes in CYP3A5 non-expressers with an unexplained correlation with MDR1 C3435T polymorphisms. Recommending routine pharmacogenetic testing of the individual SNPs in renal transplant patients is not yet feasible as the relationship of cost-effectiveness to ultimate impact on graft or patient survival must be justified by further prospective clinical trials.

Exposure to vehicle emissions results in altered blood brain barrier permeability and expression of matrix metalloproteinases and tight junction proteins in mice.

BACKGROUND: Traffic-generated air pollution-exposure is associated with adverse effects in the central nervous system (CNS) in both human exposures and animal models, including neuroinflammation and neurodegeneration. While alterations in the blood brain barrier (BBB) have been implicated as a potential mechanism of air pollution-induced CNS pathologies, pathways involved have not been elucidated. OBJECTIVES: To determine whether inhalation exposure to mixed vehicle exhaust (MVE) mediates alterations in BBB permeability, activation of matrix metalloproteinases (MMP) -2 and -9, and altered tight junction (TJ) protein expression. METHODS: Apolipoprotein (Apo) E(-/-) and C57Bl6 mice were exposed to either MVE (100 µg/m(3) PM) or filtered air (FA) for 6 hr/day for 30 days and resulting BBB permeability, expression of ROS, TJ proteins, markers of neuroinflammation, and MMP activity were assessed. Serum from study mice was applied to an in vitro BBB co-culture model and resulting alterations in transport and permeability were quantified. RESULTS: MVE-exposed Apo E(-/-) mice showed increased BBB permeability, elevated ROS and increased MMP-2 and -9 activity, compared to FA controls. Additionally, cerebral vessels from MVE-exposed mice expressed decreased levels of TJ proteins, occludin and claudin-5, and increased levels of inducible nitric oxide synthase (iNOS) and interleukin (IL)-1ß in the parenchyma. Serum from MVE-exposed animals also resulted in increased in vitro BBB permeability and altered P-glycoprotein transport activity. CONCLUSIONS: These data indicate that inhalation exposure to traffic-generated air pollutants promotes increased MMP activity and degradation of TJ proteins in the cerebral vasculature, resulting in altered BBB permeability and expression of neuroinflammatory markers.

Multiplex analysis of tumor multidrug-resistance genes expression with photonic suspension array.

An easy-operated suspension array based on silica colloidal crystal beads is developed for multiplex analysis of tumor multidrug-resistance genes expression, such as multidrug resistance 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1), and potentially single nucleotide polymorphism. In order to obtain high fluorescence intensity, controlled PCR was used to amplify targets at the samples pretreatment stage. By optimizing the conditions a hybridization procedure, which is similar to nucleic acids analysis with binary probes, was established. Small amounts of analytes 10(-19) M could be detected by the method. The K562 cell, human myeloma cell, and its multidrug-resistance string, adriamycin-selected P-glycoprotein-overexpressed K562/A02, were analyzed by using an established procedure to validate feasibility. Clinical blood samples were detected by our method and real-time PCR simultaneously to validate accuracy. Moreover, when combined with multiplex controlled PCR, the method successfully meets the requirements of multiplex analysis. Hence, the method presented is a good method for multiplex analysis of tumor multidrug-resistance genes expression.