Effect of ABCB1 most frequent polymorphisms on the accumulation of bictegravir in recombinant HEK293 cell lines.

Bictegravir, a key second-generation integrase strand transfer inhibitor in the treatment of HIV, is subject to active efflux transport mediated by ABCB1 (P-glycoprotein). Several coding variants of ABCB1 have been described and associated with variable effects on substrate drugs pharmacokinetics. Here, we investigated the effect of the four most common coding ABCB1 single nucleotide polymorphisms (i.e., c.1199G > A, c.1236C > T, c.2677G > T and c.3435C > T) on the intracellular accumulation of bictegravir. Using a previously validated HEK293 recombinant cell line model, we found decreased bictegravir intracellular concentrations in cell lines overexpressing ABCB1 as compared to control cell lines, in line with the known role of ABCB1 in bictegravir transport. However, we were unable to demonstrate any significant difference in bictegravir intracellular accumulation when comparing HEK293 cells overexpressing the wild type (1236C-2677G-3435C, 1199G) or the variant (1236C-2677G-3435T, 1236T-2677T-3435T or 1199A) proteins. These findings suggest that the ABCB1 c.1199G > A and c.1236C > T-c.2677G > T-c.3435C > T variants have no or at least limited impact on the active transport of bictegravir by ABCB1.

SEW2871 reduces seizures via the sphingosine 1-phosphate receptor-1 pathway in the pentylenetetrazol and phenobarbitone kindling model of drug-refractory epilepsy.

Epilepsy is a prevalent neurological disorder characterized by neuronal hypersynchronous discharge in the brain, leading to central nervous system (CNS) dysfunction. Despite the availability of anti-epileptic drugs (AEDs), resistance to AEDs is the greatest challenge in treating epilepsy. The role of sphingosine-1-phosphate-receptor 1 (S1PR1) in drug-resistant epilepsy is unexplored. This study investigated the effects of SEW2871, a potent S1PR1 agonist, on a phenobarbitone (PHB)-resistant pentylenetetrazol (PTZ)-kindled Wistar rat model. We measured the messenger ribonucleic acid (mRNA) expression of multi-drug resistance 1 (MDR1) and multi-drug resistance protein 5 (MRP5) as indicators for drug resistance. Rats received PHB + PTZ for 62 days to develop a drug-resistant epilepsy model. From day 48, SEW2871 (0.25, 0.5, 0.75 mg/kg, intraperitoneally [i.p.]) was administered for 14 days. Seizure scoring, behaviour, oxidative markers like reduced glutathione, catalase, superoxide dismutase, inflammatory markers like interleukin 1 beta tumour necrosis factor alpha, interferon gamma and mRNA expression (MDR1 and MRP5) were assessed, and histopathological assessments were conducted. SEW2871 demonstrated dose-dependent improvements in seizure scoring and neurobehavioral parameters with a reduction in oxidative and inflammation-induced neuronal damage. The S1PR1 agonist also downregulated MDR1 and MRP5 gene expression and significantly decreased the number of dark-stained pyknotic nuclei and increased cell density with neuronal rearrangement in the rat brain hippocampus. These findings suggest that SEW2871 might ameliorate epileptic symptoms by modulating drug resistance through downregulation of MDR1 and MRP5 gene expression.

Uricosuric Agents Affect Plasma and Kidney Concentration of Adefovir via Inhibition of Oat1 and Mrp2 in Rats.

Uricosuric agents lower serum uric acid levels by increasing urinary excretion via inhibition of urate transporter 1 (URAT1), urate reabsorption transporter in the renal proximal tubules. Probenecid and benzbromarone have been used as uricosurics, but these drugs inhibit organic anion transporters (OATs) in addition to URAT1. In this study, we investigated whether uricosuric agents interacted with adefovir, known as a substrate for OAT1, using Sprague-Dawley (SD) rats. Furthermore, involvement of other transporters, multi-drug resistance protein 2 (MRP2) in this interaction was examined using Mrp2-deficient rats. Probenecid and lesinurad increased plasma adefovir concentrations and decreased kidney-to-plasma partition coefficient (Kp) in these rats, presumably by inhibiting Oat1. Although benzbromarone had no effect on plasma adefovir concentration, it increased the Kp to 141% in SD rats. Since this effect was abolished in Mrp2-deficient rats, together with the MRP2 inhibition study, it is suggested that benzbromarone inhibits Mrp2-mediated adefovir excretion from the kidney. In contrast, dotinurad, a novel uricosuric agent that selectively inhibits URAT1, had no effect on the plasma and kidney concentrations of adefovir. Therefore, due to the lack of interaction with adefovir, dotinurad is expected to have low drug-drug interaction risk mediated by OAT1, and also by MRP2.

Intracellular Metabolomics Identifies Efflux Transporter Inhibitors in a Routine Caco-2 Cell Permeability Assay-Biological Implications.

Caco-2 screens are routinely used in laboratories to measure the permeability of compounds and can identify substrates of efflux transporters. In this study, we hypothesized that efflux transporter inhibition of a compound can be predicted by an intracellular metabolic signature in Caco-2 cells in the assay used to test intestinal permeability. Using selective inhibitors and transporter knock-out (KO) cells and a targeted Liquid Chromatography tandem Mass Spectrometry (LC-MS) method, we identified 11 metabolites increased in cells with depleted P-glycoprotein (Pgp) activity. Four metabolites were altered with Breast Cancer Resistance (BCRP) inhibition and nine metabolites were identified in the Multidrug Drug Resistance Protein 2 (MRP2) signature. A scoring system was created that could discriminate among the three transporters and validated with additional inhibitors. Pgp and MRP2 substrates did not score as inhibitors. In contrast, BCRP substrates and inhibitors showed a similar intracellular metabolomic signature. Network analysis of signature metabolites led us to investigate changes of enzymes in one-carbon metabolism (folate and methionine cycles). Our data shows that methylenetetrahydrofolate reductase (MTHFR) protein levels increased with Pgp inhibition and Thymidylate synthase (TS) protein levels were reduced with Pgp and MRP2 inhibition. In addition, the methionine cycle is also affected by both Pgp and MRP2 inhibition. In summary, we demonstrated that the routine Caco-2 assay has the potential to identify efflux transporter inhibitors in parallel with substrates in the assays currently used in many DMPK laboratories and that inhibition of efflux transporters has biological consequences.

Single nucleotide polymorphism analysis of pvmdr-1 in Plasmodium vivax isolated from military personnel of Republic of Korea in 2016 and 2017.

BACKGROUND: Malaria chemoprophylaxis using chloroquine (CQ) and primaquine (PQ) has been administered to resident soldiers in the 3rd Army of Republic of Korea (ROK) to prevent malaria infection since the year 1997. Due to mass chemoprophylaxis against malaria, concern exists about the occurrence of chloroquine resistance (CQR). This study aimed to investigate the single nucleotide polymorphisms (SNPs) of the Plasmodium vivax multi-drug resistance protein-1 (pvmdr-1) gene to monitor the risk of CQR. METHODS: SNPs of the pvmdr-1 gene were analysed in 73 soldiers of the 3rd Army of ROK diagnosed with infection by P. vivax. RESULTS: Quintuple mutations (G698S, L845F, M908L, T958M, and F1076L) were detected in 73 soldiers. A newly identified non-synonymous mutation in the Y541C position had been introduced into P. vivax malaria-endemic areas in ROK, at a frequency of 1.3% (1/73). In addition, synonymous mutations were detected at positions K44 (38.4%, 28/73), L493 (26%, 19/73), T529 (61.6%, 45/73), and E1233 (52.1%, 38/73). Based on these SNPs, pvmdr-1 sequences of ROK were classified into 6 haplotypes. The phylogenetic analysis closed to the type of North Korean showed that P. vivax malaria of ROK could be a reason of influx from North Korea. CONCLUSIONS: This study showed that synonymous and non-synonymous mutations of pvmdr-1 were observed in the malaria chemoprophylaxis-executed regions of ROK from 2016 to 2017. Based on the rapid transition of pvmdr-1 SNPs, continuous surveillance for SNPs of pvmdr-1 related to CQR in the malaria-endemic regions of ROK is essential.

Homozygous of MRP4 Gene rs1751034 C Allele Is Related to Increased Risk of Intravenous Immunoglobulin Resistance in Kawasaki Disease.

Background: Kawasaki disease (KD) is a systemic vasculitis in childhood, which mainly causes damage to coronary arteries, and intravenous immunoglobulin (IVIG) is the initial therapy. IVIG resistance increased risk of coronary complication in KD. And genetic background is involved in the occurrence of IVIG resistance. Our previous study indicated the susceptibility of Multi-drug resistance protein 4 (MRP4) SNPs to KD. This study was to clarify the relationship between MRP4 polymorphisms and IVIG resistance. Methods: We genotyped the six polymorphisms of MRP4 gene in 760 cases of KD using Taqman methods. Results: Among the six polymorphisms, only the rs1751034 polymorphism was significantly associated with IVIG resistance in KD [CC vs. TT: adjusted odds ratio (OR) = 2.54, 95% confidence interval (CI) = 1.21-5.34; CC vs. TT/TC: adjusted OR = 2.33, 95% CI = 1.12-4.83, p = 0.023]. Combined analysis of three polymorphisms indicated that patients with 3-6 risk genotypes exhibited significantly elevated risk of IVIG resistance, when compared with those with 0-2 risk genotypes (adjusted OR = 1.52, 95% CI = 1.04-2.22, p = 0.0295). Stratified analysis revealed that in term of age and gender, rs1751034 CC carriers were associated with increased risk of IVIG resistance in those aged ≤ 60 months (adjusted OR = 2.65, 95% CI = 1.23-5.71, p = 0.0133). The presence of three or more risk genotypes was significantly associated with risk of IVIG resistance in children younger than 5 years of age and males. Conclusion: Our results suggest that MRP4 rs1751034 CC is associated with increased risk of IVIG resistance in KD.

Interactions between ABCC4/MRP4 and ABCC7/CFTR in human airway epithelial cells in lung health and disease.

ATP binding cassette (ABC) transporters are present in all three domains of life - Archaea, Bacteria, and Eukarya. The conserved nature is a testament to the importance of these transporters in regulating endogenous and exogenous substrates required for life to exist. In humans, 49 ABC transporters have been identified to date with broad expression in different lung cell types with multiple transporter family members contributing to lung health and disease. The ABC transporter most commonly known to be linked to lung pathology is ABCC7, also known as cystic fibrosis transmembrane conductance regulator - CFTR. Closely related to the CFTR genomic sequence is ABCC4/multi-drug resistance protein-4. Genomic proximity is shared with physical proximity, with ABCC4 and CFTR physically coupled in cell membrane microenvironments of epithelial cells to orchestrate functional consequences of cyclic-adenosine monophosphate (cAMP)-dependent second messenger signaling and extracellular transport of endogenous and exogenous substrates. The present concise review summarizes the emerging data defining a role of the (ABCC7/CFTR)-ABCC4 macromolecular complex in human airway epithelial cells as a physiologically important pathway capable of impacting endogenous and exogenous mediator transport and ion transport in both lung health and disease.

Drug Repositioning for P-Glycoprotein Mediated Co-Expression Networks in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most fatal types of cancers that is seen in both men and women. CRC is the third most common type of cancer worldwide. Over the years, several drugs are developed for the treatment of CRC; however, patients with advanced CRC can be resistant to some drugs. P-glycoprotein (P-gp) (also known as Multidrug Resistance 1, MDR1) is a well-identified membrane transporter protein expressed by ABCB1 gene. The high expression of MDR1 protein found in several cancer types causes chemotherapy failure owing to efflux drug molecules out of the cancer cell, decreases the drug concentration, and causes drug resistance. As same as other cancers, drug-resistant CRC is one of the major obstacles for effective therapy and novel therapeutic strategies are urgently needed. Network-based approaches can be used to determine specific biomarkers, potential drug targets, or repurposing approved drugs in drug-resistant cancers. Drug repositioning is the approach for using existing drugs for a new therapeutic purpose; it is a highly efficient and low-cost process. To improve current understanding of the MDR-1-related drug resistance in CRC, we explored gene co-expression networks around ABCB1 gene with different network sizes (50, 100, 150, 200 edges) and repurposed candidate drugs targeting the ABCB1 gene and its co-expression network by using drug repositioning approach for the treatment of CRC. The candidate drugs were also assessed by using molecular docking for determining the potential of physical interactions between the drug and MDR1 protein as a drug target. We also evaluated these four networks whether they are diagnostic or prognostic features in CRC besides biological function determined by functional enrichment analysis. Lastly, differentially expressed genes of drug-resistant (i.e., oxaliplatin, methotrexate, SN38) HT29 cell lines were found and used for repurposing drugs with reversal gene expressions. As a result, it is shown that all networks exhibited high diagnostic and prognostic performance besides the identification of various drug candidates for drug-resistant patients with CRC. All these results can shed light on the development of effective diagnosis, prognosis, and treatment strategies for drug resistance in CRC.

Methods to optimize CNS exposure of drug candidates.

Among the most devastating disorders of our time, neurologic and psychiatric diseases combine to cause more disability than any other disease area. One of the key objectives within the medicinal chemistry discipline is to design molecules that penetrate into the target tissue. The objective of tissue specificity can be to gain or restrict drug access to the compartment of interest. This article briefly reviews the progress of CNS drug discovery over the past few decades. Included are the most recent efforts to harness structural and physicochemical properties assessment coupled with the impact of efflux transporters in determining brain penetration and the translation from rodent to human brain tissue targeting.

[Gadoxetic disodium-enhanced MRI to characterize T1 relaxation values and expression level of organic anion transporters and multidrug resistance protein on hepatocyte surface membrane of normal C57BL/6 mice].

Objective: The characteristics of T1 relaxation values and the expression levels of organic anion transport system (OATP) and multidrug resistance protein carrier (MRP) on hepatocyte surface membrane were quantitatively studied to evaluate liver function in normal C57BL/6 mice with gadoxetic disodium-enhanced MRI. Methods: Ten 6-weeks-old, normal C57BL/6 mice were included in this study. Gadoxetic disodium- enhanced MRI examination was performed. Longitudinal relaxation time images before and 20 min after contrast injection (hepatobiliary-specific phase) were acquired. T1-relaxation time, T1 relaxation time decline rate (△T) and rapid initial enhancement slope percentage in the first-pass study of the liver parenchyma before and after administration of gadoxetate disodium were measured. Liver parenchyma specimens were detected by Western blotting and the values ​​of OATP1, MRP2, and MRP3 were recorded. Statistical results were expressed in mean. Results: The mean T1 relaxation time of 10 normal C57BL/6 mice before and after enhancement was 659.13 ± 24.07, and 408.87 ± 27.21 ms. The mean T1 relaxation time decline rate and rapid initial enhancement slope percentage in the first-pass study was 37.12% ± 4.95% and 4.14% ± 0.96% ms. Furthermore, the mean value of OATP1, MRP2 and MRP3 were 29 952.1 ± 11 475.2, 34 376.4 ± 33 228.4 and 357 308.9 ± 64 646.5. Conclusion: T1-relaxation values, T1 relaxation time decline rate and rapid initial enhancement slope percentage in the first-pass study before and after gadoxetic disodium-enhanced MRI were determined in normal C57BL/6 mice as well as quantitative values of OATP1, MRP2 and MRP3 at the molecular level on the hepatocyte surface membrane were helpful for liver injury model with control study.

Gadoxetic disodium-enhanced MRI to characterize T1 relaxation values and expression level of organic anion transporters and multidrug resistance protein on hepatocyte surface membrane of normal C57BL/6 mice / 中华肝脏病杂志

Objective@#The characteristics of T1 relaxation values and the expression levels of organic anion transport system (OATP) and multidrug resistance protein carrier (MRP) on hepatocyte surface membrane were quantitatively studied to evaluate liver function in normal C57BL/6 mice with gadoxetic disodium-enhanced MRI.@*Methods@#Ten 6-weeks-old, normal C57BL/6 mice were included in this study. Gadoxetic disodium- enhanced MRI examination was performed. Longitudinal relaxation time images before and 20 min after contrast injection (hepatobiliary-specific phase) were acquired. T1-relaxation time, T1 relaxation time decline rate (△T) and rapid initial enhancement slope percentage in the first-pass study of the liver parenchyma before and after administration of gadoxetate disodium were measured. Liver parenchyma specimens were detected by Western blotting and the values ​​of OATP1, MRP2, and MRP3 were recorded. Statistical results were expressed in mean.@*Results@#The mean T1 relaxation time of 10 normal C57BL/6 mice before and after enhancement was 659.13 ± 24.07, and 408.87 ± 27.21 ms. The mean T1 relaxation time decline rate and rapid initial enhancement slope percentage in the first-pass study was 37.12% ± 4.95% and 4.14% ± 0.96% ms. Furthermore, the mean value of OATP1, MRP2 and MRP3 were 29 952.1 ± 11 475.2, 34 376.4 ± 33 228.4 and 357 308.9 ± 64 646.5.@*Conclusion@#T1-relaxation values, T1 relaxation time decline rate and rapid initial enhancement slope percentage in the first-pass study before and after gadoxetic disodium-enhanced MRI were determined in normal C57BL/6 mice as well as quantitative values of OATP1, MRP2 and MRP3 at the molecular level on the hepatocyte surface membrane were helpful for liver injury model with control study.

[Effect of different concentrations of Gegen Qinlian decoction on expression of P-glycoprotein and multi-drug resistance protein transporter in Caco-2 cells].

To investigate the effects of Gegen Qinlian decoction on the expression of P-glycoprotein (P-gp) and multi-drug resistance protein (MRP) in epithelial cells of human colon adenocarcinoma Caco-2 cells．The effects of different concentrations of Gegen Qinlian decoction on the expression levels of p-gp and MRP1-6 mRNA in Caco-2 cells were detected by real time quantitative reverse transcriptase PCR (qRT-PCR).12 h after drug treatment (5.00 g·L⁻¹), the expression levels of MDR1 and MRP1-6 were significantly down-regulated at concentration of 5.00 g·L⁻¹; the mRNA expression levels of MDR1，MRP1，MRP2，MRP4，MRP5 and MRP6 were significantly down-regulated at concentration of 2.50 g·L⁻¹; only the expression levels of MRP2 and MRP5 were significantly affected at concentration of 1.00 g·L⁻¹. The results showed that the expression levels of MDR1 and MRP1-6 mRNA in Caco-2 cells could be down-regulated in a dose-dependent manner. Gegen Qinlian decoction may reduce drug efflux by down-regulating the mRNA expression of cell transporters in Caco-2 cell, and increase the time of drug action, thereby enhancing the bioavailability of chemotherapeutic drugs.

Activation of soluble guanylyl cyclase with inhibition of multidrug resistance protein inhibitor-4 (MRP4) as a new antiplatelet therapy.

The intracellular levels of cyclic GMP are controlled by its rate of formation through nitric oxide-mediated stimulation of soluble guanylate cyclase (sGC) and its degradation by phosphodiesterases. Multidrug resistance protein 4 (MRP4) expressed in human platelets pumps cyclic nucleotides out of cells. In search for new antiplatelet strategies, we tested the hypothesis that sGC activation concomitant with MRP4 inhibition confers higher antiplatelet efficacy compared with monotherapy alone. This study was undertaken to investigate the pharmacological association of the sGC activator BAY 60-2770 with the MRP4 inhibitor MK571 on human washed platelets. Collagen- and thrombin-induced platelet aggregation and ATP-release reaction assays were performed. BAY 60-2770 (0.001-10 µM) produced significant inhibitions of agonist-induced platelet aggregation accompanied by reduced ATP-release. Pre-incubation with 10 µM MK571 alone had no significant effect on platelet aggregation and ATP release, but it produced a left displacement by about of 10-100-fold in the concentration-response curves to BAY 60-2770. Pre-incubation with MK571increased and decreased, respectively, the intracellular and extracellular levels of cGMP to BAY 60-2770, whereas the cAMP levels remained unchanged. The increased VASP-serine 239 phosphorylation in BAY 60-2770-treated platelets was enhanced by MK571. In Fluo-4-loaded platelets, BAY 60-2770 reduced the intracellular Ca2+ levels, an effect significantly potentiated by MK571. Flow cytometry assays showed that BAY 60-2770 reduces the αIIbß3 integrin activation, which was further reduced by MK571 association. Blocking the MRP4-mediated efflux of cGMP may be a potential mechanism to enhance the antiplatelet efficacy of sGC activators.

An animal's sex influences the effects of the excipient PEG 400 on the intestinal P-gp protein and mRNA levels, which has implications for oral drug absorption.

There is a growing body of evidence which suggests that formerly regarded "inert" pharmaceutical excipients have the potential to influence oral drug bioavailability. The solubilizing agent polyethylene glycol 400 (PEG 400), for instance, has a sex-specific effect on P-glycoprotein (P-gp)-mediated drug bioavailability. We hypothesized that such an effect could be via PEG-induced alteration of P-gp activity and/or expression to different extents in males and females. To test this hypothesis in vivo, we investigated the influence of orally administered PEG 400 on the protein content and mRNA expression of P-gp in different regions of the gastrointestinal tract in male and female rats. Fasted rats received an oral dose of PEG 400 and at different time intervals, rats were sacrificed and their intestines were collected. The P-gp protein and mRNA expression in different intestinal segments (duodenum, jejunum, ileum and colon) were measured by Western blotting and PCR, respectively. It was found that P-gp protein and mRNA levels increased along the gastrointestinal tract in control animals (i.e. without PEG administration), and was higher in males compared to the female rats. The oral administration of PEG 400 decreased the P-gp expression in the jejunum, ileum and colon of males but not in the corresponding segments in females. This sex-dependent influence of PEG 400 on P-gp levels reflects and explains the sex-related effect of PEG 400 on oral absorption of certain drugs. The data further adds to the growing literature on the importance of taking into consideration an individual's sex for optimal drug administration.

Effect of different concentrations of Gegen Qinlian decoction on expression of P-glycoprotein and multi-drug resistance protein transporter in Caco-2 cells / 中国中药杂志

To investigate the effects of Gegen Qinlian decoction on the expression of P-glycoprotein (P-gp) and multi-drug resistance protein (MRP) in epithelial cells of human colon adenocarcinoma Caco-2 cells．The effects of different concentrations of Gegen Qinlian decoction on the expression levels of p-gp and MRP1-6 mRNA in Caco-2 cells were detected by real time quantitative reverse transcriptase PCR (qRT-PCR).12 h after drug treatment (5.00 g·L⁻¹), the expression levels of MDR1 and MRP1-6 were significantly down-regulated at concentration of 5.00 g·L⁻¹; the mRNA expression levels of MDR1，MRP1，MRP2，MRP4，MRP5 and MRP6 were significantly down-regulated at concentration of 2.50 g·L⁻¹; only the expression levels of MRP2 and MRP5 were significantly affected at concentration of 1.00 g·L⁻¹. The results showed that the expression levels of MDR1 and MRP1-6 mRNA in Caco-2 cells could be down-regulated in a dose-dependent manner. Gegen Qinlian decoction may reduce drug efflux by down-regulating the mRNA expression of cell transporters in Caco-2 cell, and increase the time of drug action, thereby enhancing the bioavailability of chemotherapeutic drugs.

Quantitative Evaluation of Drug Resistance Profile of Cells Expressing Wild-Type or Genetic Polymorphic Variants of the Human ABC Transporter ABCC4.

Broad-spectrum resistance in cancer cells is often caused by the overexpression of ABC transporters; which varies across individuals because of genetic single-nucleotide polymorphisms (SNPs). In the present study; we focused on human ABCC4 and established cells expressing the wild-type (WT) or SNP variants of human ABCC4 using the Flp-In™ system (Invitrogen, Life Technologies Corp, Carlsbad, CA, USA) based on Flp recombinase-mediated transfection to quantitatively evaluate the effects of nonsynonymous SNPs on the drug resistance profiles of cells. The mRNA levels of the cells expressing each ABCC4 variant were comparable. 3-(4,5-Dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay clearly indicated that the EC50 values of azathioprine against cells expressing ABCC4 (WT) were 1.4-1.7-fold higher than those against cells expressing SNP variants of ABCC4 (M184K; N297S; K304N or E757K). EC50 values of 6-mercaptopurine or 7-Ethyl-10-hydroxy-camptothecin (SN-38) against cells expressing ABCC4 (WT) were also 1.4-2.0- or 1.9-fold higher than those against cells expressing the SNP variants of ABCC4 (K304N or E757K) or (K304N; P403L or E757K); respectively. These results indicate that the effects of nonsynonymous SNPs on the drug resistance profiles of cells expressing ABCC4 can be quantitatively evaluated using the Flp-In™ system.

Excipient-mediated alteration in drug bioavailability in the rat depends on the sex of the animal.

The pharmaceutical excipient, polyethylene glycol 400 (PEG 400), unexpectedly alters the bioavailability of the BCS class III drug ranitidine in a sex-dependent manner. As ranitidine is a substrate for the efflux transporter P-glycoprotein (P-gp), we hypothesized that the sex-related influence could be due to interactions between PEG 400 and P-gp. In this study, we tested this hypothesis by: i) measuring the influence of PEG 400 on the oral bioavailability of another P-gp substrate (ampicillin) and of a non-P-gp substrate (metformin); and ii) measuring the effect of PEG 400 on drug bioavailability in the presence of a P-gp inhibitor (cyclosporine A) in male and female rats. We found that PEG 400 significantly increased (p<0.05) the bioavailability of ampicillin (the P-gp substrate) in male rats, but not in female ones. In contrast, PEG 400 had no influence on the bioavailability of the non-P-gp substrate, metformin in male or female rats. Inhibition of P-gp by oral pre-treatment with cyclosporine A increased the bioavailability of the P-gp substrates (ampicillin and ranitidine) in males and females (p<0.05), and to a greater extent in males, but had no influence on the bioavailability of metformin in either male or female rats. These results prove the hypothesis that the sex-specific effect of PEG 400 on the bioavailability of certain drugs is due to the interaction of PEG 400 with the efflux transporter P-gp.

5-HT2A receptor SPECT imaging with [¹²³I]R91150 under P-gp inhibition with tariquidar: More is better?

INTRODUCTION: Pharmacological P-glycoprotein (P-gp) inhibition with tariquidar (TQD) is considered a promising strategy for the augmentation of radiotracer brain uptake. However, a region-dependent effect may compromise the robustness of quantitative studies. For this reason, we studied the effect of a TQD pretreatment on 5-HT2A imaging with [(123)I]R91150 and compared results with those obtained in Mdr1a knock-out (KO) rats. METHODS: Ex vivo autoradiography was performed in TQD (15 mg/kg) pretreated wild-type (WT-TQD), Mdr1a knock-out (KO) and untreated WT rats for Specific Binding Ratio (SBR) estimation. In vivo dynamic SPECT imaging with serial arterial blood sampling was performed in the former two groups of rats and kinetic analysis was performed with a one tissue-compartment (1TC) model and the Specific Uptake Ratio (SUR). Results were analyzed statistically using repeated measures ANOVA. RESULTS: SBR values differed between WT-TQD, Mdr1a KO and WT rats in a region-dependent manner (p<0.0001). In vivo brain uptake of radiotracer did not differ between groups. Similarly, kinetic analysis provided distribution volume (V(T)) values that did not differ significantly between groups. SUR binding potential (BPND) values from both groups highly correlated with corresponding V(T) (r=0.970, p<0.0001 and r=0.962, p<0.0001, respectively). However, SUR measured over averaged images between 100 and 120 min, using cerebellum as reference region, demonstrated values that were, by average, 2.99±0.53 times higher in the WT-TQD group, with the difference between groups being region-dependent (p<0.001). In addition, coefficient of variation of the SUR BPND values across brain regions was significantly higher in the WT-TQD rats (41.25%±9.63% versus 11.13%±5.59%, p<0.0001). CONCLUSION: P-gp inhibition with TQD leads to region-dependent effect in the rat brain, with probably sub-optimal effect in cerebellum. This warrants attention when it is used as a reference region for quantitative studies.

Chemoresistance of multi-drug resistance malignant glioma cells mediated by DNA-PKcs and its molecular mechanism / 吉林大学学报(医学版)

Objective:To obesrve the influence of DNA-PKcs in the chemoresistance of multi-drug resistance malignant glioma cells,and to explore its molecuIar mechanism in chemoresistance.Methods:siRNA was used to construct the DNA-PKcs knockdown human glioma U251 cell line;Western blotting method was used to detect the expressions of DNA-PKcs in U251 cells (U251 cells), doxorubicin (ADM)resistant U251 cells (U251/ADM cells),DNA-PKcs knockdown and ADM resistant U251 cells (U251/ADM/siDNA-PKcs cells).CCK8 method was used to detect the cell proliferation activity in three groups;Western blotting method was used to detect the expressions of MDR1, pNF-κB/p6, total Akt, pAkt/T308 and pAKT/S473 in the cells in three groups. Results:The expression level of DNA-PKcs in U251/ADM cells was significantly higher than those in U251 cells and U251/ADM/siDNA-PKcs cells (P 0.05).Conclusion:DNA-PKcs can significantly enhance the chemoresistance of multi-drug resistance malignant glioma cells,the underlying mechanism is related to up-regulation of pAKT/S473,pNF-κB/p65 and MDR1 expressions.

Lipid flopping in the liver.

Bile is synthesized in the liver and is essential for the emulsification of dietary lipids and lipid-soluble vitamins. It is a complex mixture of amphiphilic bile acids (BAs; which act as detergent molecules), the membrane phospholipid phosphatidylcholine (PC), cholesterol and a variety of endogenous metabolites and waste products. Over the last 20 years, the combined effort of clinicians, geneticists, physiologists and biochemists has shown that each of these bile components is transported across the canalicular membrane of the hepatocyte by its own specific ATP-binding cassette (ABC) transporter. The bile salt export pump (BSEP) ABCB11 transports the BAs and drives bile flow from the liver, but it is now clear that two lipid transporters, ABCB4 (which flops PC into the bile) and the P-type ATPase ATP8B1/CDC50 (which flips a different phospholipid in the opposite direction) play equally critical roles that protect the biliary tree from the detergent activity of the bile acids. Understanding the interdependency of these lipid floppases and flippases has allowed the development of an assay to measure ABCB4 function. ABCB4 harbours numerous mis-sense mutations which probably reflects the spectrum of liver disease rooted in ABCB4 aetiology. Characterization of the effect of these mutations at the protein level opens the possibility for the development of personalized prognosis and treatment.