Efflux Transporter P-glycoprotein Research Articles

An isogenic neurovascular unit model comprised of human induced pluripotent stem cell-derived brain microvascular endothelial cells, pericytes, astrocytes, and neurons

BackgroundBrain microvascular endothelial cells (BMECs) astrocytes, neurons, and pericytes form the neurovascular unit (NVU). Interactions with NVU cells endow BMECs with extremely tight barriers via the expression of tight junction proteins, a host of active efflux and nutrient transporters, and reduced transcellular transport. To recreate the BMEC-enhancing functions of NVU cells, we combined BMECs, astrocytes, neurons, and brain pericyte-like cells.MethodsBMECs, neurons, astrocytes, and brain like pericytes were differentiated from human induced pluripotent stem cells (iPSCs) and placed in a Transwell-type NVU model. BMECs were placed in co-culture with neurons, astrocytes, and/or pericytes alone or in varying combinations and critical barrier properties were monitored.ResultsCo-culture with pericytes followed by a mixture of neurons and astrocytes (1:3) induced the greatest barrier tightening in BMECs, supported by a significant increase in junctional localization of occludin. BMECs also expressed active P-glycoprotein (PGP) efflux transporters under baseline BMEC monoculture conditions and continued to express baseline active PGP efflux transporters regardless of co-culture conditions. Finally, brain-like pericyte co-culture significantly reduced the rate of non-specific transcytosis across BMECs.ConclusionsImportantly, each cell type in the NVU model was differentiated from the same donor iPSC source, yielding an isogenic model that could prove enabling for enhanced personalized modeling of the NVU in human health and disease.

Demonstrating suitability of the Caco-2 cell model for BCS-based biowaiver according to the recent FDA and ICH harmonised guidelines.

According to the regulatory guidelines, one of the critical steps in using in-vitro permeability methods for permeability classification is to demonstrate the suitability of the method. Here, suitability of the permeability method by using a monolayer of cultured epithelial cells was verified with different criteria. Imaging with a transmission electron microscope was used for characterisation of the cells. Monolayer integrity was confirmed by transepithelial electrical resistance measurements and permeability of zero permeability marker compounds. Real-time polymerase chain reaction was employed to evaluate expression levels of 84 known transporters. Samples for bidirectional permeability determination were quantified by ultra-performance liquid chromatography. The Caco-2 cells grow in an intact monolayer and morphologically resemble enterocytes. Genes of 84 known transporters were expressed at different levels; furthermore, expression was time depended. Functional expression of efflux transporter P-glycoprotein was confirmed. We established a correlation between permeability coefficients of 21 tested drug substances ranging from low, moderate and high absorption with human fraction absorbed literature data (R2 =0.84). Assay standardisation assures the consistency of experimental data. Only such fully characterised model has the ability to accurately predict drug's intestinal permeability at the early stage of research or for the BCS-based biowaiver application.

Comparative pharmacokinetics of tedizolid in rat plasma and cerebrospinal fluid

To investigate the possibility of tedizolid phosphate's application in the treatment of intracranial infection, a preclinical comparative pharmacokinetic study was designed. Based on the assumption that the classic efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) may participate in the transportation of TDZ, two groups of rats were intravenously administered 6 mg/kg tedizolid phosphate alone or 6 mg/kg tedizolid phosphate combined with 1 mg/kg elacridar which was an inhibitor of P-gp and BCRP. Plasma and cerebrospinal fluid samples were collected according to a pharmacokinetic schedule. All the plasma and cerebrospinal fluid samples were assessed with a validated LC-MS/MS method. The penetration ratio of tedizolid from the blood to cerebrospinal fluid was calculated, and a comparison of the penetration ratios between the two groups was made. The mean Cmax of tedizolid in the CSF in the tedizolid phosphate group and the tedizolid phosphate combined with elacridar group was 154 ng/mL and 300 ng/mL, respectively, and the mean penetration ratio of tedizolid in the tedizolid phosphate group and the tedizolid phosphate combined with elacridar group was 2.16% and 3.53%, respectively. The relatively high Cmax in the CSF proved the possibility of tedizolid phosphate's application in the treatment of intracranial infection, and the higher penetration ratios, Cmax, csf and AUCcsf of the rats in co-administered elacridar group than those in the single-administration group indicated that the transporters P-gp and BCRP might be involved in the transportation of tedizolid.

Kurkumin Meningkatkan Sensitivitas Sel Kanker Payudara terhadap Tamoksifen melalui Penghambatan Ekspresi P-glikoprotein dan Breast Cancer Resistance Protein

The decreasing of sensitivity or resistance to tamoxifen occured after long-term treatment in breast cancer. One of the major factor in tamoxifen resistance is over expression of efflux transporter P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP). Curcumin has known as inhibitor of P-gp and BCRP. The addition of curcumin to the tamoxifen resistant cells is expected to increase the sensitivity of breast cancer cells to tamoxifen. This study aim to know the effect of curcumin in increasing the cell sensitivity to tamoxifen through inhibition of P-gp and BCRP transporter efflux. MCF-7 breast cancer cell line was induced with tamoxifen 1 µM for 10 passage (MCF-7(T)), then cell viability and mRNA expression of P-gp and BCRP were analyzed. To the MCF-7(T) cells, curcumin was given at of 5/10/20 µM with or without tamoxifen for 5 days and cell viability and mRNA expression of P-gp and BCRP were analyzed on day 5th. As positive control, verapamil 50 µM was used as P-gp inhibitor, ritonavir 15 µM and nelfinavir 15 µM were used as BCRP inhibitor. The results showed that MCF-7(T) cells sensitivity to tamoxifen decreased with 11.8 times, the cell viability increased 10.82 fold and mRNA expression of P-gp and BCRP increased 4.04 fold. Then after administration of curcumin with or without tamoxifen for 5 days, the cell viability and the mRNA expression of P-gp and BCRP decreased. As conclusion, curcumin increased the sensitivity of MCF-7(T) to tamoxifen characterized by the decreasing of cell viability and mRNA expression of P-gp and BCRP. However, the administration of combination of curcumin with tamoxifen was more potent than just curcumin. The increased sensitivity was estimated at least partly through the inhibition of P-gp and BCRP mRNA expression by curcumin

The Effects of P‐glycoprotein Inhibition on Norbuprenorphine‐induced Neonatal Abstinence Syndrome (NAS)

Treating pregnant, opioid‐addicted women with the opioid buprenorphine improves mother‐child outcomes, but can cause neonatal abstinence syndrome (NAS), a potentially life‐threatening withdrawal syndrome newborns often develop following chronic prenatal exposure to opioids. NAS severity is independent of maternal buprenorphine dose, suggesting that inter‐individual variance in the pharmacokinetics of buprenorphine may influence NAS. We previously showed that prenatal exposure to the major active metabolite of buprenorphine, norbuprenorphine (NorBUP), can induce NAS. NorBUP is a substrate of the highly polymorphic placental efflux transporter P‐glycoprotein, which transports its substrates from fetal to maternal circulation. We tested the hypothesis that inhibiting P‐glycoprotein increases NorBUP‐induced NAS severity. We administered a subthreshold dose of NorBUP or vehicle to pregnant rats from gestation day (GD) 9 until post‐delivery and treated them twice daily with the p‐glycoprotein inhibitor Elacridar or vehicle on GD18‐21. After delivery, pups were observed for precipitated withdrawal signs following administration of naltrexone or saline. NorBUP and Elacridar together, but not individually, increased naltrexone‐precipitated withdrawal signs, providing the first evidence that P‐glycoprotein activity can substantially modulate the effects of NorBUP on the fetus and may therefore contribute to the uncoupling of maternal buprenorphine dose and NAS severity.Support or Funding InformationThe project described was supported by a Summer Undergraduate Research Fellowship from the American Society for Pharmacology and Experimental Therapeutics, the UAMS Fellow‐to‐Faculty Award (Arkansas Biomedical Institute, Tobacco Settlement Funds), a UAMS Research Scholar Pilot Grant Award in Child Health, and the Translational Research Institute (TRI), grants 1U54TR001629‐01A1 and KL2TR000063 through the National Center for Advancing Translational Sciences of the National Institutes of Health (NIH). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regional difference in intestinal drug absorption as a measure for the potential effect of P-glycoprotein efflux transporters.

The aim of this research was to assess regional difference in the intestinal absorption of ranitidine HCl as an indicator for the potential effect of P-glycoprotein (P-gp) efflux transporters. In situ rabbit intestinal perfusion was used to investigate absorption of ranitidine HCl, a substrate for P-gp efflux from duodenum, jejunum, ileum and colon. This was conducted both in the presence and absence of piperine as P-gp inhibitor. Ranitidine HCl was incompletely absorbed from rabbit intestine. The length normalized absorptive clearance (PeA/L) of ranitidine HCl was ranked as colon>duodenum>jejunum>ileum. This is the reverse order of the magnitude of P-gp expression. Coperfusion of piperine with ranitidine HCl significantly increased the PeA/L of ranitidine HCl from jejunum and ileum with no significant change on the absorption from duodenum and colon. This was confirmed by significant reduction in the length required for complete ranitidine HCl absorption from jejunum and ileum in presence piperine. The results indicate that P-gp transporters play a major role in determining regional difference in intestinal absorption of ranitidine HCl. Thus, the regional absorption of drugs may be taken as an indirect indication for the role of P-gp in intestinal absorption.

Potential of Natural-Based Anticancer Compounds for P-Glycoprotein Inhibition.

Medicinal value of natural products comes from symbiotic and competitive evolution in Earth's complex biosphere. Billions of years of co-evolutionary interactions among millions of species have produced a large repertoire of defense molecules effective in fighting bacteria, viral, and fungal pathogens. Each species contains millions of different and useful molecules and new research technologies enabled the screening of molecules and complex mixtures from diverse biological sources. Traditional use of plants and other species led to the discovery of many bioactive compounds with various properties. In the last four decades, a large number of them were evaluated for their potential to treat cancer. Penetration of drugs into the cancer cell is necessary for their lethal pharmacological effect through interaction with intracellular target molecules. Increased activity of membrane efflux pumps reduces the intracellular drug accumulation, thereby preventing drug-target interactions. The discovery of the efflux transporter P-glycoprotein (P-gp) in multidrug resistant (MDR) cancer cells prompted the efforts in overcoming drug resistance by P-gp inhibition. The search for nontoxic anticancer agents from natural sources able to overcome MDR has been imperative in the field of drug design and discovery. Herein, we review various natural compounds from diverse sources emphasizing their potential to inhibit P-gp activity and/or expression.

TGF-β1 elevates P-gp and BCRP in hepatocellular carcinoma through HOTAIR/miR-145 axis.

Multidrug resistance (MDR) is common in patients and has been linked to transforming growth factor-β1 (TGF-β1) and overexpression of drug efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), although the molecular mechanisms remain largely unknown. This study aimed to investigate the mechanisms underlying TGF-β1-induced MDR in hepatocellular carcinoma (HCC) cells. It was found that TGF-β1 upregulated HOX transcript antisense RNA (HOTAIR) expression in HCC cells. When drosophila mothers against decapentaplegic 4 (SMAD4) was silenced, HOTAIR expression was accordingly reduced. Meanwhile, miR-145 expression was increased in the case HOTAIR was silenced. If the enhancer of zeste homolog 2 (EZH2) was knocked down using small interfering RNA (siRNA), miR-145 expression was decreased. Then, the regulatory role of miR-145 in P-gp and BCRP expression was explored. The results showed that the expression of P-gp and BCRP protein was suppressed by miR-145 through binding to the 3'-untranslated regions (3'-UTRs) of P-gp and BCRP. In conclusion, our study revealed a novel mechanism explaining TGF-β1-induced MDR in HCC through upregulating P-gp and BCRP via the SMAD4/HOTAIR/miR-145 axis.

Oral coadministration of elacridar and ritonavir enhances brain accumulation and oral availability of the novel ALK/ROS1 inhibitor lorlatinib

Lorlatinib, a novel generation oral anaplastic lymphoma kinase (ALK) and ROS1 inhibitor with high membrane and blood-brain barrier permeability, recently received accelerated approval for treatment of ALK-rearranged non-small-cell lung cancer (NSCLC), and its further clinical development is ongoing. We previously found that the efflux transporter P-glycoprotein (MDR1/ABCB1) restricts lorlatinib brain accumulation and that the drug-metabolizing enzyme cytochrome P450-3A (CYP3A) limits its oral availability. Using genetically modified mouse models, we investigated the impact of targeted pharmacological inhibitors on lorlatinib pharmacokinetics and bioavailability. Upon oral administration of lorlatinib, the plasma AUC0-8h in CYP3A4-humanized mice was ∼1.8-fold lower than in wild-type and Cyp3a–/– mice. Oral coadministration of the CYP3A inhibitor ritonavir caused reversion to the AUC0-8h levels seen in wild-type and Cyp3a–/– mice, without altering the relative tissue distribution of lorlatinib. Moreover, simultaneous pharmacological inhibition of P-glycoprotein and CYP3A4 with oral elacridar and ritonavir in CYP3A4-humanized mice profoundly increased lorlatinib brain concentrations, but not its oral availability or other relative tissue distribution. Oral lorlatinib pharmacokinetics was not significantly affected by absence of the multispecific Oatp1a/1b drug uptake transporters. The absolute oral bioavailability of lorlatinib over 8 h in wild-type, Cyp3a–/–, and CYP3A4-humanized mice was 81.6%, 72.9%, and 58.5%, respectively. Lorlatinib thus has good oral bioavailability, which is markedly restricted by human CYP3A4 but not by mouse Cyp3a. Pharmacological inhibition of CYP3A4 reversed these effects, and simultaneous P-gp inhibition with elacridar boosted absolute brain levels of lorlatinib by 16-fold without obvious toxicity. These insights may help to optimize the clinical application of lorlatinib.

Formulation and Evaluation of Propranolol HCl Floating Tablets-A Gastro Retentive Drug Delivery

The objective of present study was to develop a gastroretentive drug delivery system of propranolol hydrochloride. The biggest problem in oral drug delivery is low and erratic drug bioavailability. This study” shown the formulation of floating tablets of Propranolol HCl by employing carbopol and HPMC as polymers. One of the disadvantages in using propranolol is extensive first pass metabolism of drug and only 25% reaches systemic circulation. The bioavailability of propranolol increases in presence of food. Also, the absorption of various drugs such as propranolol through P-glycoprotein (P-gp) efflux transporter is low and erratic. The density of P-gp increases toward the distal part of the gastrointestinal tract (GIT). Therefore, it was decided to formulate floating tablet of propranolol so that it remains in the upper part of GIT for longer time. The prepared tablets were evaluated among all the formulations (P1-P5). Formulations were evaluated for floating lag time, duration of floating, dimensional stability, drug content and in vitro drug release profile. It was observed that formulation”-5 has shown better buoyancy and dissolution profile.

A new LDL-masked liposomal doxorubicin overcomes drug resistance in osteosarcoma

One of the chemotherapeutic drugs in the first-line treatment of osteosarcoma is doxorubicin (dox). However, its anticancer efficacy is limited by the overexpression of the multidrug efflux transporter P-glycoprotein (Pgp) which extrudes doxorubicin, disrupts drug accumulation and thus hinders the cytotoxic activity of dox The drug extrusion activity of Pgp is enhanced by the presence of cholesterol within the plasma membrane. Pgp expression is up-regulated by multiple transcription factors including hypoxia inducible factor-1α (HIF-1α). Decreasing Pgp expression and drug efflux activity may enhance the efficacy of doxorubicin against drug resistant osteosarcoma. To achieve this goal, we treated two human dox-sensitive cell lines (U-2OS and Saos-2), and their sublines displaying increasing Pgp levels and dox resistance, with a non-toxic dose of atorvastatin. This statin was able to decrease the biosynthesis of farnesyl pyrophosphate (FPP) and cholesterol, two key metabolites that were markedly increased in drug resistant sublines. By reducing the FPP levels, atorvastatin decreased the Ras/ERK1/2/HIF-1α axis and down-regulated Pgp expression. Moreover, by decreasing cholesterol biosynthesis atorvastatin increased the levels of low density lipoprotein receptor (LDLR), that was also progressively increased in the dox-resistant sublines. Taking advantage of this findings, we treated dox-resistant osteosarcoma with atorvastatin followed by treatment with dox encapsulated in liposomes decorated with an LDLR-binding peptide, hence facilitating binding to, and endocytosis via LDLR. This treatment strategy induced high retention of dox in dox-resistant cells, reduced cell viability in vitro and displayed growth inhibition of dox-resistance in mouse xenograft model. Hence, we propose the combination of statins plus LDL-decorated liposomal dox as a novel treatment strategy for Pgp-expressing multidrug resistant osteosarcomas.

Diffuse Alveolar Hemorrhage Secondary to Direct Oral Anticoagulants: A Systematic Review of Reported Cases

Introduction:Direct oral anticoagulants (DOACs), also known as non-vitamin K oral anticoagulants, include rivaroxaban, dabigatran, apixiban, edoxaban, and betrixaban. Guidelines have recently identified DOACs as the first line treatment in venous thromboembolism and nonvalvular atrial fibrillation. As the use of these anticoagulants has gradually increased in last few years, the reports of bleeding-related adverse drug events with these agents has increased. There are very few data related to diffuse alveolar hemorrhage (DAH) with DOACs, a dreaded complication of any anticoagulant.Objective:To perform a systematic review of all reported cases of DAH secondary to DOAC use.Methods:Using several search terms, we reviewed all cases of DAH associated with DOACs which were published and indexed in English language in PubMed and EMBASE in the last 10 years. The bibliography of each relevant article was searched for additional related reports. Statistical analysis was performed using Statistical Packages for the Social Science (SPSS).Results:A total of 34 cases were included in the study. Mean age was 69±15 years; 68% were male, 32% were female. Rivaroxaban, dabigatran, apixaban, and edoxaban were used in 41%, 35%, 21%, 3% of patients respectively. Atrial fibrillation was the commonest indication for DOAC use, 68%. Hemoptysis was the commonest presenting symptom, 74%. DAH was diagnosed with Computed Tomography (CT), Bronchoalveolar Lavage (BAL), and both in 68%, 56%, and 35% respectively. A total of 18 (53%) cases required mechanical ventilation. There was a total of 7 (21%) deaths, while 20 (59%) survived.Conclusion:Despite reports of lower risk of major bleeding compared to other anticoagulants, DAH is still a possibility with DOAC use. DOACs' absorption and elimination relies largely on P-glycoprotein (P-gp) efflux transporters, coadministration of P-gp inhibitors such as Amiodarone, a common antiarrhythmic used in atrial fibrillation in conjunction with anticoagulation, may result in disturbance of DOAC's concentrations in the blood, increasing the risk of potential side effects. Autoimmune diseases can lead to DAH independently. Owing to the diseased lung tissue in patients with autoimmune disease, the use of DOAC's may enhance the already present risk of DAH. Renal function plays a role in excretion of DOAC's, AKI may cause accumulation of DOAC's in the body leading to major side effects. Newer cases reported the use of idracuzimab; a newly FDA-approved drug for dabigatran reversal. In conclusion; caution should be taken with the increasing use of DOAC's in management of complex care patients requiring anticoagulation, prompt diagnosis and management is key for survival and prevention of complications, and new reversal agents may play a future role in the management of such complications. DisclosuresNo relevant conflicts of interest to declare.

Increasing intratumor C/EBP-\u03b2 LIP and nitric oxide levels overcome resistance to doxorubicin in triple negative breast cancer

BackgroundTriple negative breast cancer (TNBC) easily develops resistance to the first-line drug doxorubicin, because of the high levels of the drug efflux transporter P-glycoprotein (Pgp) and the activation of pro-survival pathways dependent on endoplasmic reticulum (ER). Interfering with these mechanisms may overcome the resistance to doxorubicin, a still unmet need in TNBC.MethodsWe analyzed a panel of human and murine breast cancer cells for their resistance to doxorubicin, Pgp expression, lysosome and proteasome activity, nitrite production, ER-dependent cell death and immunogenic cell death parameters. We evaluated the efficacy of genetic (C/EBP-β LIP induction) and pharmacological strategies (lysosome and proteasome inhibitors), in restoring the ER-dependent and immunogenic-dependent cell death induced by doxorubicin, in vitro and in syngeneic mice bearing chemoresistant TNBC. The results were analyzed by one-way analysis of variance test.ResultsWe found that TNBC cells characterized by high levels of Pgp and resistance to doxorubicin, had low induction of the ER-dependent pro-apoptotic factor C/EBP-β LIP upon doxorubicin treatment and high activities of lysosome and proteasome that constitutively destroyed LIP. The combination of chloroquine and bortezomib restored doxorubicin sensitivity by activating multiple and interconnected mechanisms. First, chloroquine and bortezomib prevented C/EBP-β LIP degradation and activated LIP-dependent CHOP/TRB3/caspase 3 axis in response to doxorubicin. Second, C/EBP-β LIP down-regulated Pgp and up-regulated calreticulin that triggered the dendritic cell (DC)-mediated phagocytosis of tumor cell, followed by the activation of anti-tumor CD8+T-lymphocytes upon doxorubicin treatment. Third, chloroquine and bortezomib increased the endogenous production of nitric oxide that further induced C/EBP-β LIP and inhibited Pgp activity, enhancing doxorubicin’s cytotoxicity. In orthotopic models of resistant TNBC, intratumor C/EBP-β LIP induction - achieved by a specific expression vector or by chloroquine and bortezomib - effectively reduced tumor growth and Pgp expression, increased intra-tumor apoptosis and anti-tumor immune-infiltrate, rescuing the efficacy of doxorubicin.ConclusionsWe suggest that preventing C/EBP-β LIP degradation by lysosome and proteasome inhibitors triggers multiple virtuous circuitries that restore ER-dependent apoptosis, down-regulate Pgp and re-activate the DC/CD8+T-lymphocytes response against TNBC. Lysosome and proteasome inhibitors associated with doxorubicin may overcome the resistance to the drug in TNBC.

Breast cancer resistance protein is the enemy of hypericin accumulation and toxicity of hypericin-mediated photodynamic therapy

Breast cancer resistance protein (BCRP) belongs to the family of ATP-binding cassette (ABC) transporters, overexpression of which can confer a multidrug-resistant phenotype in cancer cells and tumors. BCRP mediates efflux of numerous xenobiotics, including various chemotherapeutic agents and photosensitizers. Hypericin (HY) is a naturally-occurring photosensitizer synthesized by plants of the genus Hypericum. Our recently published results indicate that accumulation of HY in cancer cells of different tissue origin can be affected mostly by BCRP. Considering all known facts, the main goal of this study was to verify whether not only HY accumulation but also toxicity of HY-mediated photodynamic therapy (PDT) can be affected by the presence of some ABC transporters. To specifically prove our hypothesis, we used an experimental model of human leukemia cell lines differing in the expression level of the main drug efflux transporters P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and BCRP. The lowest HY accumulation, and consequently the highest resistance to HY-PDT, was found in cells overexpressing BCRP. Moreover, pretreatment with BCRP inhibitor Ko143 significantly increased HY accumulation and sensitized cells to HY-PDT. Therefore, our findings represent direct evidence that BCRP is the nemesis of HY accumulation and toxicity of HY-PDT. Thus, we should emphasize that individualized screening for BCRP expression and activity may represent a useful tool for prediction of HY-mediated photodynamic diagnosis (PDD) or PDT effectiveness.

Assessment of P-glycoprotein gene expression in adult stage of Haemonchus contortus in vivo exposed to ivermectin

The efflux transporter P-glycoprotein (P-gp) has been implicated in multidrug resistance of different nematode parasites affecting livestock species. Increased expression of P-gp in nematodes after their in vitro as well as in vivo exposure to anthelmintics suggests a role of P-gp in drug resistance. The current study evaluated the P-gp gene expression in a highly-resistant isolate of the sheep nematode Haemonchus contortus, selected after exposure to ivermectin (IVM) treatments at 10-fold the therapeutic dose. Four lambs were artificially infected with L3 (7000 L3/animal) of a previously selected IVM highly resistant H. contortus isolate. Forty five (45) days after infection, adult worms were collected at 0 (untreated), 6, 12 and 24 h post-oral IVM (2 mg/kg) administration. The relative transcription levels of different H. contortus P-gp genes were studied by quantitative real-time PCR (qPCR) and confirmed by RNA-seq. P-gp1 and P-gp11 gene expressions did not change throughout the experimental sampling period. P-gp3 and P-gp9.1 transcripts decreased significantly at both 12 and 24 h post IVM exposure. P-gp2 expression was progressively increased in a time-dependent manner at 1.81 (6 h), 2.08 (12 h) and 2.49 (24 h)-fold compared to adult worms not exposed (control 0 h) to IVM, although without reaching statistically significant differences (P > 0.05). P-gp12 was neither detected by qPCR nor by RNA-seq analysis. These relatively modest changes in the P-gp gene expression could not be enough to explain the high level of IVM resistance displayed by the H. contortus isolate under assessment. Overexpression of membrane drug transporters including P-gp has been associated with IVM resistance in different nematode parasites. However, some evidences suggest that resistance to IVM and other macrocyclic lactones may develop by multiple mechanisms. Further studies are needed to improve the understanding of resistance mechanisms in adult stages of H. contortus.

Lipomers (Lipid-polymer Hybrid Particles) of Vardenafil Hydrochloride: a Promising Dual Platform for Modifying the Drug Release Rate and Enhancing Its Oral Bioavailability.

Vardenafil hydrochloride is commonly used for the curing of erectile dysfunction. VAR suffers certain limitations: (i) short elimination half-life (4-5h), (ii) low aqueous solubility (0.11mg/mL), (iii) susceptibility to extensive first-pass metabolism and drug efflux transporters (P-glycoprotein), and (iv) limited (15%) oral bioavailability. The current study focused on the development of VAR lipomers as promising modified release systems able to enhance oral bioavailability. VAR-lipomers (lipid-polymer complexes) were successfully developed by a modified precipitation technique employing a lipid (polyglyceryl-6-distearate or glyceryl tristearate) and an amphiphilic polymer (Gantrez®). Three VAR:lipid ratios [1:1, 1:2, and 1:3] and three VAR:Gantrez® ratios [4:1, 2:1, and 1:1] were investigated. Solid-state characterization studies involved differential scanning calorimetry (DSC), X-ray diffraction (XRD), and Fourier-transform infrared (FT-IR) spectroscopy. The systems were assessed for particle size, polydispersity index (PDI), zeta-potential, VAR entrapment-efficiency (EE%), morphology, and VAR released % after 2h (Q2h) and 8h (Q8h). The best-achieved system (the highest desirability) was promoted for pharmacokinetic studies in fasted rabbits. Statistical analysis of data revealed that L9 system (PGDS, VAR, and Gantrez®; 3:1:1, respectively) had the highest desirability (0.85) with respect to spherical particle size (622.15nm), PDI (0.11), zeta-potential (-27.90mV), EE% (62.80%), Q2h (43.45%), and Q8h (77.40%). With respect to Levitra® tablets, the significantly higher relative bioavailability (170%), delayed Tmax, and extended MRT(0-∞) clarified the dual ability of L9 system. Lipomers are emerging systems capable of modifying the rate of VAR release and promoting its oral bioavailability.

Promoter Haplotypes of the ABCB1 Gene Encoding the P-Glycoprotein Differentially Affect Its Promoter Activity by Altering Transcription Factor Binding.

Promoter single nucleotide polymorphisms (SNPs) of the ABCB1 gene, encoding the placental efflux transporter P-glycoprotein, can alter its expression and affect fetal exposure to therapeutics and environmental xenobiotics. SNPs are not arrayed as independent variants but as combinations forming defined haplotypes. Recently, we defined the haplotypes encompassing ABCB1 promoter SNPs and found that ABCB1 haplotypes differentially affect its promoter activity. The mechanism(s) by which ABCB1 haplotypes alter its promoter activity are not known. We hypothesize that the haplotype-dependent differences in ABCB1 promoter activity are due to haplotype-specific alterations in transcription factor (TF) binding. To test our hypothesis, we used a TF binding profile array and determined whether differences in TF binding exist across different ABCB1 haplotypes. TFs showing significant haplotype binding differences were mechanistically evaluated using small interfering RNA (siRNA) in cultured human placental cells. Our data indicate significant haplotype-dependent differences in TF binding. Our siRNA studies showed that the regulatory effects of TFs on promoter activity are also haplotype dependent. Our data provide a mechanistic explanation for the differential effects of ABCB1 haplotypes on its promoter activity and underscore the importance of evaluating genetic variants in the context of haplotypes rather than individual SNPs when investigating their effects on gene/protein expression and disease risk.

Bisphenol A (BPA) and bisphenol S (BPS) alter the promoter activity of the ABCB1 gene encoding P-glycoprotein in the human placenta in a haplotype-dependent manner

Exposure to bisphenols (BPA and BPS) during pregnancy can significantly affect fetal development and increase risk of adverse health consequences, however the underlying mechanisms are not fully elucidated. In human placenta, the efflux transporter P-glycoprotein (P-gp), encoded by the ABCB1 gene, extrudes its substrates from the trophoblasts back into the maternal circulation. Alterations in levels of placental P-gp could therefore significantly affect fetal exposure to xenobiotics that are P-gp substrates. The ABCB1 promoter contains many single nucleotide polymorphisms (SNPs). In the genome, SNPs are not arrayed as independent variants but as combinations forming defined haplotypes. Recently, we determined the haplotype sequences encompassing the ABCB1 promoter SNPs and found that promoter haplotypes differentially affect ABCB1 promoter activity. Here we investigate the effect of BPA and BPS on ABCB1 promoter activity by testing the hypothesis that BPA and BPS exposure affect ABCB1 promoter activity in a haplotype-dependent manner. Our data indicate that acute exposure to 50 nM BPA induced a significant haplotype-dependent increase in ABCB1 promoter activity (P < .05). However, acute exposure to 0.5 nM BPS induced a significant decrease (P < .05) in promoter activity that was haplotype-dependent. Chronic exposure to BPA and BPS individually (5 nM and 0.3 nM, respectively) or as a mixture (5 nM BPA:1.5 nM BPS) induced significant haplotype-dependent increases (P < .01) in ABCB1 promoter activity. Our data indicate that BPA and BPS significantly alter ABCB1 promoter activity in a haplotype- and exposure type- dependent manners. Such alteration could significantly impact placental P-gp levels and alter fetal exposure to many therapeutic and environmental xenobiotics.

Rate and extent of mitragynine and 7-hydroxymitragynine blood-brain barrier transport and their intra-brain distribution: the missing link in pharmacodynamic studies.

Mitragyna speciosa is reported to be beneficial for the management of chronic pain and opioid withdrawal in the evolving opioid epidemic. Data on the blood-brain barrier (BBB) transport of mitragynine and 7-hydroxymitragynine, the active compounds of the plant, are still lacking and inconclusive. Here, we present for the first time the rate and the extent of mitragynine and 7-hydroxymitragynine transport across the BBB, with an investigation of their post-BBB intra-brain distribution. We utilized an in vitro BBB model to study the rate of BBB permeation of the compounds and their interaction with efflux transporter P-glycoprotein (P-gp). Mitragynine showed higher apical-to-basolateral (A-B, i.e. blood-to-brain side) permeability than 7-hydroxymitragynine. 7-Hydroxymitragynine showed a tendency to efflux, with efflux ratio (B-A/A-B) of 1.39. Both were found to inhibit the P-gp and are also subject to efflux by the P-gp. Assessment of the extent of BBB transport in vivo in rats from unbound brain to plasma concentration ratios (Kp,uu,brain ) revealed extensive efflux of both compounds, with less than 10 percent of unbound mitragynine and 7-hydroxymitragynine in plasma crossing the BBB. By contrast, the extent of intra-brain distribution was significantly different, with mitragynine having 18-fold higher brain tissue uptake in brain slice assay compared with 7-hydroxymitragynine. Mitragynine showed a moderate capacity to accumulate inside brain parenchymal cells, while 7-hydroxymitragynine showed restricted cellular barrier transport. The presented findings from this systematic investigation of brain pharmacokinetics of mitragynine and 7-hydroxymitragynine are essential for design and interpretation of in vivo experiments aiming to establish exposure-response relationship.

CYP3A5*3 and ABCB1 61A>G Significantly Influence Dose-adjusted Trough Blood Tacrolimus Concentrations in the First Three Months Post-Kidney Transplantation.

Tacrolimus (TAC) is a first-line immunosuppressant used to prevent organ rejection after kidney transplantation. There is large inter-individual variability in its pharmacokinetics. Single nucleotide polymorphisms (SNPs) in genes encoding TAC metabolizing enzymes cytochromes P450 3A4/5 (CYP3A4/5), P-glycoprotein efflux transporter (ABCB1), their expression regulator pregnane X receptor (NR1I2) and CYP3A co-factor cytochrome P450 reductase (POR) have been studied for their effects on tacrolimus disposition. However, except for CYP3A5*3, controversies remain about their roles in predicting dose-adjusted trough blood TAC concentrations (C0 /D). This study aimed to investigate the effects of ABCB1 (61A>G, 1199G>A, 1236C>T, 2677G>T and 3435C>T), CYP3A4*22, CYP3A5*3, NR1I2 (8055C>T, 63396C>T and -25385C>T) and POR*28 SNPs on TAC C0 /D. In total, 165 kidney transplant recipients were included in this study. SNPs were genotyped by probe-based real-time polymerase chain reaction. Associations between log-transformed whole blood TAC C0 /D (measured at 1 and 3 months post-transplant) and genotypes/haplotypes were assessed by linear mixed effects analysis, controlling for age, sex and haematocrit. It was observed that CYP3A5 expressors (*1/*1 + *1/*3) (p = 5.5 × 10-16 ) and ABCB1 61G allele carriers (p = 0.001) had lower log-transformed TAC C0 /D (56% and 26% lower geometric mean TAC C0 /D, respectively) and accounted for approximately 30% and 4%, respectively, of log-transformed TAC C0 /D variability in the first 3 months post-transplant. In conclusion, CYP3A5*3 is a major, and ABCB1 61A>G is a novel, although minor, genetic factor affecting TAC C0 /D in kidney transplant recipients.