Changes In P-glycoprotein Research Articles

Study on the reversal of epileptic drug resistance by tetramethylpyrazine in combination with carbamazepine through modulation of P-Glycoprotein expression in rat brain microvessel endothelial cell.

The purpose of this study was to detect the changes of P-Glycoprotein (P-GP) expression in rat brain microvessel endothelial cell line RBE4 after the action of Tetramethylpyrazine (TMP) on Carbamazepine (CBZ), so as to clarify the potential mechanism of TMP combined with CBZ against intractable epilepsy drug resistance. The RBE4 cell line was utilized for in vitro analysis. Cells were divided into control, CBZ, and CBZ-TMP group. The expression of P-GP was assessed using Western blot and reverse transcription polymerase chain reaction (RT-PCR). Intracellular concentration of CBZ was measured through high-performance liquid chromatography (HPLC). The differential expression of mRNA was evaluated by RNA sequencing. The intracellular concentration of CBZ in the CBZ-TMP group was significantly higher than that in other groups. The expression of P-GP in the CBZ group was significantly higher than that in the control group, while in the CBZ&TMP group, it was significantly lower than that in the other groups. Comparative analysis also revealed some differentially expressed genes. Compared with the CBZ group, FAM106A, SLC3A2, TENM2, etc. were upregulated most significantly in the CBZ&TMP group. ZBTB10, WDR7, STARD13, etc. were downregulated most significantly. Results suggest that TMP increases the intracellular concentration of CBZ, downregulates the expression of P-GP increased by CBZ, and modulates related cellular metabolism and signaling pathways, thus reversing the drug resistance mechanism of intractable epilepsy, providing a theoretical basis for the combination of traditional Chinese medicine and antiepileptic drugs.

Precision Analysis of Drug-Induced Conformational Changes of P-Glycoprotein Observed in AFM

P-glycoprotein (Pgp) is an ATP-Binding Cassette transporter commonly overexpressed in cancer cells. This protein effluxes drugs out of the cell, leading to multi-drug resistance. Significant research interest surrounds the determination of the mechanism of action. A powerful single-molecule tool is the Atomic Force Microscope (AFM), which can directly image biomolecules in physiological buffer and temperature. Pgp reconstituted in supported lipid bilayers is imaged in the presence of cancer drugs and ATP-analogs to probe conformational changes. The high spatial resolution of the AFM must be matched by the precision of the post-imaging analysis. Previously, we have introduced the Hessian blob algorithm as a method of particle detection that minimizes user input [B.P. Marsh et al. Scientific Reports 8, 978 (2018)]. With the addition of scale-space Laplace interpolation to detect background levels, we increase the precision of particle heights, revealing previously unresolved populations of Pgp. Model selection techniques such as the Bayesian information criterion add to the objectivity of data interpretation. Together, these analyses refine our ability to deduce ligand-induced conformational shifts of Pgp in an unbiased manner.

Physicochemical and biopharmaceutical characterization of novel Matrix-Liposomes

Matrix-Liposomes (MLs) are a very promising solid oral drug delivery system; however, data on their interaction with biological membranes are not available. Here, we describe the quality of MLs manufactured by dual centrifugation. MLs were prepared with a Z-average range of 139 to 160 nm and a PDI of 0.18 to 0.25. To investigate the effect of MLs on intestinal tissue (with and without mucolytic treatment), we then established an ex vivo rat intestine model. The integrity of the epithelial membranes of rat intestine was not affected by the incubation with MLs without or with pre-mucolytic treatment. Tissue samples were also analysed for changes in P-glycoprotein (P-gp) expression and function. The net secretion of the P-gp substrate Rh123 across the rat duodenum was increased in the presence of MLs. To summarize, MLs do not affect intestinal epithelial integrity, although they impact Rh123 secretion. In future, these novel MLs have to be further evaluated for proficient intestinal drug delivery.

Direct Observation of Ligand-induced Conformational Changes of P-glycoprotein in AFM and Correlation with Surface-absorbed Activity Measurements

P-glycoprotein (Pgp) is an ATP-binding cassette transporter that is capable of pumping drugs out of a cell. This membrane protein is overexpressed in cancer cells and has been identified as a major cause of cancer drug failure. Hence, significant interest surrounds determining the structure/function relationship of this protein. Atomic Force Microscopy (AFM) is a single molecule method that can probe conformational dynamics in biologically relevant conditions and is well suited for membrane protein studies. In previous work [K.P. Sigdel et al. Biochemical Pharmacology 156, 302 (2018)], we directly imaged Pgp in supported lipid bilayers and observed structural changes of specific sides of the protein, distinguished by antibodies and the inherent asymmetry in the bilayer. Here, we extend the study to conformational dynamics in the presence of ligands including drugs and ATP analogs. We also introduce glass as a supporting surface in lieu of mica as evidence suggests [K. Chattrakun et al. Langmuir 35, 12246 (2019)] that this rougher surface can provide a more native-like environment for membrane proteins. Independent of surface species selection, absorption can raise questions about activity. An ATP hydrolysis assay tracking the release of radiolabeled gamma-phosphate was performed on the surface-bound Pgp system. Taken together, the data suggest that hydrolysis activity is maintained for our AFM preparations.

INFLUENCE OF VILDAGLIPTIN AND GLIQUIDONE COMBINATION ON P-GLYCOPROTEIN FUNCTIONAL ACTIVITY AND EXPRESSION ON THE BACKGROUND OF NORM AND IN EXPERIMENTAL ALLOXAN-INDUCED DIABETES MELLITUS TYPE 2

In the in vivo study on rabbits investigated the effect of the vildagliptin and gliqui-done combination on the background of norm and experimental alloxan-induced diabetes mellitus (DM) type 2 on the P-glycoprotein (P-gp, ABCB1 protein) functional activity and its expression in the tissues of the jejunum, liver, kidney and the blood-brain barrier. Introduction of combination of vildagliptin at a dose of 5 mg/kg and gliquidone 10 mg/kg body weight on the background of norm for 14 days did not result in P-glycoprotein changes. On the 5-day of the cancellation of this combination observed inhibition of transporter protein functional activity without altering its expression. In the combination therapy of vildagliptin and gliquidone alloxan-induced diabetes type 2 showed complete recovery of P-glycoprotein functional activity and expression reduced on the pathology background. On the 5-day of treatment withdrawal again occurred inhibition of functional activity of the transporter protein and a decrease in its expression.

An automated method measures variability in P-glycoprotein and ABCG2 densities across brain regions and brain matter

Changes in P-glycoprotein and ABCG2 densities may play a role in amyloid-beta accumulation in Alzheimer’s disease. However, previous studies report conflicting results from different brain regions, without correcting for changes in vessel density. We developed an automated method to measure transporter density exclusively within the vascular space, thereby correcting for vessel density. We then examined variability in transporter density across brain regions, matter, and disease using two cohorts of post-mortem brains from Alzheimer’s disease patients and age-matched controls. Changes in transporter density were also investigated in capillaries near plaques and on the mRNA level. P-glycoprotein density varied with brain region and matter, whereas ABCG2 density varied with brain matter. In temporal cortex, P-glycoprotein density was 53% lower in Alzheimer’s disease samples than in controls, and was reduced by 35% in capillaries near plaque deposits within Alzheimer’s disease samples. ABCG2 density was unaffected in Alzheimer’s disease. No differences were detected at the transcript level. Our study indicates that region-specific changes in transporter densities can occur globally and locally near amyloid-beta deposits in Alzheimer’s disease, providing an explanation for conflicting results in the literature. When differences in region and matter are accounted for, changes in density can be reproducibly measured using our automated method.

Cholesterol-dependent conformational changes of P-glycoprotein are detected by the 15D3 monoclonal antibody

The 15D3 mouse monoclonal antibody (mAb) binds an uncharacterized extracellular epitope of the ATP Binding Cassette (ABC) transporter human P-glycoprotein (Pgp). Depletion of cell plasma membrane cholesterol by using methyl-β-cyclodextrin or other chemically modified β-cyclodextrins decreased the Pgp binding affinity of 15D3 mAb. UIC2 mAb, which is known to distinguish two conformers of this ABC transporter, binds only a fraction of cell surface Pgps. UIC2 mAb non-reactive pools of Pgp can be identified with other extracellular mAbs such as 15D3. Cyclosporin A (CsA) can shift non-reactive Pgps into their UIC2-reactive conformation: a phenomenon called the “UIC2 shift”. Competition studies proposed these two mAbs share overlapping epitopes and can reveal conformational changes of Pgp that correlate (r=0.97) with the cholesterol content of cells. An apparent increase in competition of these mAbs suggested a conformational change similar to those found in the presence of CsA. However, the reason turned out not to be the UIC2-shift because cholesterol removal from the plasma membrane (PM) reduced the amount of detectable Pgps by 15D3 mAb. This study showed that 15D3 mAb bound to a conformation sensitive epitope of Pgp that was responsive to PM cholesterol levels. These conformational changes were gradual and not as great as the changes observed between the two conformers recognized by the UIC2 mAb.

Comparative pharmacokinetics of the main compounds of Shanzhuyu extract after oral administration in normal and chronic kidney disease rats

Ethnopharmacological relevancePharmacokinetic studies on traditional Chinese medicine are useful to evaluate and predict the drug efficacy and safety. The renal impairment may affect drug clearance and other pharmacokinetic processes which can increase toxicity and drug to drug interactions or cause ineffective therapy. Pharmacokinetic studies in pathological status rats might be meaningful for revealing the action mechanism and improving clinical medication of the herb medicine. Materials and methodsA highly sensitive and rapid ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) method with multiple-reaction monitoring (MRM) mode was developed and validated for simultaneous quantitation of morroniside and loganin in normal and doxorubicin-induced chronic kidney disease (CKD) rat plasma after oral administration of Shanzhuyu (fruit of Cornus officinalis) extract. ResultsBoth calibration curves gave satisfactory linearity (r>0.99) at linear range of 1.96–1962.5ngmL−1 for morroniside, 1.53–1531.25ngmL−1 for loganin. The precision and accuracy of the in vivo study were assessed by intra-day and inter-day assays. The percentages of relative standard deviation (RSD) were all within 9.58% and the accuracy (RE) was in the −6.02% to 8.11% range. The extraction recoveries of morroniside, loganin and internal standard (IS) were all >67.62% and the matrix effects ranged from 95.07% to 102.75%. ConclusionsThe pharmacokinetic behavior of morroniside and loganin in normal and CKD rat plasma was determined in this paper. The significant different pharmacokinetic parameters might partly result from the changes of P-glycoprotein and metabolic enzymes in the pathological state. The pharmacokinetic research in the pathological state might provide more useful information to guide the clinical usage of the herb medicine.

Rewired Metabolism in Drug-resistant Leukemia Cells: A METABOLIC SWITCH HALLMARKED BY REDUCED DEPENDENCE ON EXOGENOUS GLUTAMINE

Cancer cells that escape induction therapy are a major cause of relapse. Understanding metabolic alterations associated with drug resistance opens up unexplored opportunities for the development of new therapeutic strategies. Here, we applied a broad spectrum of technologies including RNA sequencing, global untargeted metabolomics, and stable isotope labeling mass spectrometry to identify metabolic changes in P-glycoprotein overexpressing T-cell acute lymphoblastic leukemia (ALL) cells, which escaped a therapeutically relevant daunorubicin treatment. We show that compared with sensitive ALL cells, resistant leukemia cells possess a fundamentally rewired central metabolism characterized by reduced dependence on glutamine despite a lack of expression of glutamate-ammonia ligase (GLUL), a higher demand for glucose and an altered rate of fatty acid β-oxidation, accompanied by a decreased pantothenic acid uptake capacity. We experimentally validate our findings by selectively targeting components of this metabolic switch, using approved drugs and starvation approaches followed by cell viability analyses in both the ALL cells and in an acute myeloid leukemia (AML) sensitive/resistant cell line pair. We demonstrate how comparative metabolomics and RNA expression profiling of drug-sensitive and -resistant cells expose targetable metabolic changes and potential resistance markers. Our results show that drug resistance is associated with significant metabolic costs in cancer cells, which could be exploited using new therapeutic strategies.

FTY720 enhances chemosensitivity of colon cancer cells to doxorubicin and etoposide via the modulation of P-glycoprotein and multidrug resistance protein 1.

This study aimed to investigate the effects of FTY720 on inducing cell growth inhibition and enhancing the cytotoxicity of anti-cancer drugs in the human colon cancer cell line HCT-8 and its multidrug-resistant cell line HCT-8/5-fluorouracil (HCT-8/5-Fu). Cell viability and apoptosis after being treated with FTY720 alone or in combination with doxorubicin (DOX) and etoposide (VP16) were tested in HCT-8 and HCT-8/5-Fu cells. The changes in P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1) were determined at the mRNA and functional levels. FTY720 showed anti-proliferative activity against cancer cells in a dose-dependent and time-dependent manner and could enhance the cytotoxicity of DOX and VP16 in both HCT-8 and HCT-8/5-Fu cell lines. In addition, treatment with FTY720 resulted in the promotion of VP16-induced cell apoptosis and an increased accumulation of intracellular DOX and two specific fluorescent substrates of P-gp and MRP1 through the inhibition of efflux and the suppression of gene expression. FTY720 exerts its chemosensitization effect in HCT-8 and HCT-8/5-Fu cell lines by promoting cell apoptosis and inhibiting P-gp and MRP1, which could be applied as a potential co-adjuvant therapeutic modality.

Altered GABAA Receptor Density and Unaltered Blood–Brain Barrier Transport in a Kainate Model of Epilepsy: An In Vivo Study Using 11C-Flumazenil and PET

The aim of the present study was to investigate if flumazenil blood-brain barrier transport and binding to the benzodiazepine site on the γ-aminobutyric acid A (GABA(A)) receptor complex is altered in an experimental model of epilepsy and subsequently to study if changes in P-glycoprotein (P-gp)-mediated efflux of flumazenil at the blood-brain barrier may confound interpretation of (11)C-flumazenil PET in epilepsy. The transport of flumazenil across the blood-brain barrier and the binding to the benzodiazepine site on the GABA(A) receptors in 5 different brain regions was studied and compared between controls and kainate-treated rats, a model of temporal lobe epilepsy, with and without tariquidar pretreatment. In total, 29 rats underwent 2 consecutive (11)C-flumazenil PET scans, each one lasting 30 min. The tracer was mixed with different amounts of isotopically unmodified flumazenil (4, 20, 100, or 400 μg) to cover a wide range of receptor occupancies during the scan. Before the second scan, the rats were pretreated with a 3 or 15 mg/kg dose of the P-gp inhibitor tariquidar. The second scan was then obtained according to the same protocol as the first scan. GABA(A) receptor density, B(max), was estimated as 44 ± 2 ng x mL(-1) in the hippocampus and as 33 ± 2 ng x mL(-1) in the cerebellum, with intermediate values in the occipital cortex, parietal cortex, and caudate putamen. B(max) was decreased by 12% in kainate-treated rats, compared with controls. The radiotracer equilibrium dissociation constant, K(D), was similar in both rat groups and all brain regions and was estimated as 5.9 ± 0.9 ng x mL(-1). There was no difference in flumazenil transport across the blood-brain barrier between control and kainate-treated rats, and the effect of tariquidar treatment was similar in both rat groups. Tariquidar treatment also decreased flumazenil transport out of the brain by 73%, increased the volume of distribution in the brain by 24%, and did not influence B(max) or K(D), compared with baseline. B(max) was decreased in kainate-treated rats, compared with controls, but no alteration in the blood-brain barrier transport of flumazenil was observed. P-gp inhibition by tariquidar treatment increased brain concentrations of flumazenil in both groups, but B(max) estimates were not influenced, suggesting that (11)C-flumazenil scanning is not confounded by alterations in P-gp function.

(R)-[11C]verapamil PET studies to assess changes in P-glycoprotein expression and functionality in rat blood-brain barrier after exposure to kainate-induced status epilepticus.

BackgroundIncreased functionality of efflux transporters at the blood-brain barrier may contribute to decreased drug concentrations at the target site in CNS diseases like epilepsy. In the rat, pharmacoresistant epilepsy can be mimicked by inducing status epilepticus by intraperitoneal injection of kainate, which leads to development of spontaneous seizures after 3 weeks to 3 months. The aim of this study was to investigate potential changes in P-glycoprotein (P-gp) expression and functionality at an early stage after induction of status epilepticus by kainate.Methods(R)-[11C]verapamil, which is currently the most frequently used positron emission tomography (PET) ligand for determining P-gp functionality at the blood-brain barrier, was used in kainate and saline (control) treated rats, at 7 days after treatment. To investigate the effect of P-gp on (R)-[11C]verapamil brain distribution, both groups were studied without or with co-administration of the P-gp inhibitor tariquidar. P-gp expression was determined using immunohistochemistry in post mortem brains. (R)-[11C]verapamil kinetics were analyzed with approaches common in PET research (Logan analysis, and compartmental modelling of individual profiles) as well as by population mixed effects modelling (NONMEM).ResultsAll data analysis approaches indicated only modest differences in brain distribution of (R)-[11C]verapamil between saline and kainate treated rats, while tariquidar treatment in both groups resulted in a more than 10-fold increase. NONMEM provided most precise parameter estimates. P-gp expression was found to be similar for kainate and saline treated rats.ConclusionsP-gp expression and functionality does not seem to change at early stage after induction of anticipated pharmacoresistant epilepsy by kainate.

Abstract 3636: The complex mechanism of resistance of human neuroblastoma cells to chemotherapeutic drugs

Abstract It has become evident that the biological processes which allow malignant cells to survive in the presence of cytotoxic drugs are extraordinarily complex, and recent findings indicate that a multitude of cellular defense mechanisms may be involved in the development of drug resistance. In order to dissect the dynamic changes in gene expression during the process of acquiring drug resistance, four human neuroblastoma cell lines (IMR 32, BE(2)-C, SK-N-SH and SH-SY5Y) were used to generate drug-resistant cell lines using clinically appropriate chemotherapeutic agents (Doxorubicin and Busulfan). Additionally, in order to understand the physiological role of histone deacetylase (HDACs) in the development of drug resistance in human neuroblastoma and to determine if this development can be delayed or totally suppressed by the HDACs inhibition, the cells were separated into those that were pretreated with a histone deacetylase (HDAC) inhibitor: suberoylanilide hydroxamic acid (SAHA), and those that were not pretreated. Cells were deemed drug-resistant when the cells were able to grow efficiently in drug concentrations of two log concentrations higher than the IC50 level. Neuroblastoma cells were collected at each log phase increase of cytotoxic agent to compare changes in P-glycoprotein and midkine expression; cross-resistance between chemotherapeutic agents, and presence of survival proteins in each group. The creation of these resistant human neuroblastoma cell lines and the determination of the transcriptional changes occurring as the cells become resistant may have important implications for clinical treatment of human neuroblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3636.

Van't Hoff Analysis of ATP Binding to Human P-glycoprotein (ABCB1)

ATP binding to ABC transport proteins is an established step in the mechanism by which substrates are transported. Basal ATPase activity (activity seen in the absence of transported drugs) also facilitates multidrug transport by ABC proteins (Al-Shawi et al. 2003, J. Biol. Chem. 278, 52629). Therefore, we aimed to develop a non-perturbing method for detecting structural changes of P-glycoprotein associated with ATP binding during turnover. To accomplish this we analyzed heat capacity changes during ATP binding to P-glycoprotein by generating van't Hoff relationships for KMATP values for ATP binding and KiAMPPNP values for AMPPNP binding (non-hydrolysable analog of ATP).The rate of ATP hydrolysis by purified P-glycoprotein was measured as a function of ATP and AMPPNP concentrations at different temperatures from 23 to 35°C, in the presence or absence of saturating concentration of drug. Plots of Ln(KaATP) as a function of reciprocal absolute temperature in the presence of verapamil, valinomycin, colchicine and in absence of drug were fit by a nonlinear van't Hoff equation. Changes in heat capacity were derived from the fits. For basal activity, the Ln(KaATP) increased with reciprocal temperature. The heat capacity change was positive. In the presence of verapamil the relationship declined and the heat capacity change was negative. Results obtained with the other drugs were similar. This suggests that P-glycoprotein became more hydrated on ATP binding during basal activity and adopted a more open form. In contrast, in the presence of verapamil P-glycoprotein became less hydrated and adopted a more compact form. Overall, the data support our contention that there are two structurally distinct reaction pathways for ATP hydrolysis by ABC transporters and further help clarify the coupling mechanism of ATP hydrolysis to substrate transport. Supported by NIH grant GM52502.

A clinical-scale selective allodepletion approach for the treatment of HLA-mismatched and matched donor-recipient pairs using expanded T lymphocytes as antigen-presenting cells and a TH9402-based photodepletion technique

AbstractSelective allodepletion is a strategy to eliminate host-reactive donor T cells from hematopoietic stem cell allografts to prevent graft-versus-host disease while conserving useful donor immune functions. To overcome fluctuations in activation-based surface marker expression and achieve a more consistent and effective allodepletion, we investigated a photodepletion process targeting activation-based changes in p-glycoprotein that result in an altered efflux of the photosensitizer TH9402. Expanded lymphocytes, generated using anti-CD3 and IL-2, were cocultured with responder cells from HLA-matched or -mismatched donors. Optimal results were achieved when cocultured cells were incubated with 7.5 μM TH9402, followed by dye extrusion and exposure to 5 Joule/cm2 light energy at 5 × 106 cells/mL. In mismatched stimulator-responder pairs, the median reduction of alloreactivity was 474-fold (range, 43-fold to 864-fold) compared with the unmanipulated responder. Third-party responses were maintained with a median 1.4-fold (range, 0.9-fold to 3.3-fold) reduction. In matched pairs, alloreactive helper T-lymphocyte precursors were reduced to lower than 1:100 000, while third-party responses remained higher than 1:10 000. This establishes a clinical-scale process capable of highly efficient, reproducible, selective removal of alloreactive lymphocytes from lymphocyte transplant products performed under current Good Manufacturing Practice. This procedure is currently being investigated in a clinical trial of allotransplantation.

Nucleotide-induced Structural Changes in P-glycoprotein Observed by Electron Microscopy

P-glycoprotein (Pgp) is an ATP hydrolysis driven multidrug efflux pump, which, when overexpressed in the plasma membrane of certain cancers, can lead to the failure of chemotherapy. Previously, we have presented a projection structure of nucleotide-free mouse Pgp from electron microscopic images of lipid monolayer-generated two-dimensional crystals ( Lee, J. Y., Urbatsch, I. L., Senior, A. E., and Wilkens, S. (2002) J. Biol. Chem. 277, 40125-40131 ). Here we have analyzed the structure of cysteine-free human Pgp from two-dimensional crystals that were generated with the same lipid-monolayer technique in the absence and presence of various nucleotides. The images show that human Pgp has a similar structure to the mouse protein. Furthermore, the analysis of projection structures obtained under different nucleotide conditions suggests that Pgp can exist in at least two major conformations, one of which shows a central cavity between the N- and C-terminal halves of the molecule and another in which the two halves have moved sideways, thereby closing the central cavity. Intermediate conformations were observed for some nucleotide/vanadate combinations. A low-resolution, three-dimensional model of human Pgp was calculated from tilted specimen crystallized in the presence of the non-hydrolyzable nucleotide analog, adenosine 5'-O-(thiotriphosphate). The structural analysis presented here adds to the emerging picture that multidrug ABC transporters function by switching between two major conformations in a nucleotide-dependent manner.

Assessment of Ileal Epithelial P-Glycoprotein Dysfunction Induced by Ischemia/Reperfusion using in vivo Animal Model

We presented the ischemia/reperfusion (I/R) model which can evaluate changes in P-glycoprotein (P-gp) function induced by lipid peroxidation using surgical-sutures connected with the spring balance. The superior mesenteric artery and vein was occluded by hanging itself using surgical-sutures connected with the spring balance for 60 min (ischemia), followed by reperfusion by cutting of sutures. To determine the hanging force of blood vessel during ischemia, treatment at the hanging force of 50g load, 100g load and 150g load for 60 min was carried out and survival rate was evaluated. Although our 150g load group had some effect on survival, the survival was 100% in the case of 50g and 100g load groups. Thiobarbituric acid-reactive substance (TBA-RS) as an indicator of lipid peroxidation and P-gp expression level after I/R was increased and decreased in a load-dependent manner during ischemia, respectively. Also, the decrease in the level of mdr1a mRNA and function of P-gp by I/R depended on load during ischemia. The changes in TBA-RS, P-gp expression level and P-gp function observed in this study corresponded with our in vitro I/R model reported previously. In conclusion, it was shown that this in vivo I/R model can evaluate the function of P-gp through lipid peroxidation.

P-glycoprotein selection in strains of Haemonchus contortus resistant to benzimidazoles

Anthelmintic resistance in parasitic nematodes of livestock is a chronic problem in many parts of the world. Benzimidazoles are effective, broad-spectrum anthelmintics that bind to and selectively depolymerise microtubules. Resistance to the benzimidazoles, however, developed quickly and is caused by genetic changes in genes encoding β-tubulins, subunits of microtubules. In Haemonchus contortus, resistance to avermectins has been correlated with genetic changes at a gene encoding a P-glycoprotein, a cell membrane transport protein that has a very high affinity for ivermectin. The substrate specificity of P-glycoprotein is very broad, and resistance to benzimidazoles can be modulated by lectins specific for P-glycoprotein. We investigated the possibility that genetic changes in P-glycoprotein might be correlated with benzimidazole resistance in nematodes. An analysis of restriction fragment length polymorphisms of a P-glycoprotein gene from a sensitive and a cambendazole-selected strain of H. contortus, derived from the sensitive strain, showed a significant difference in allele frequencies between strains. The frequency of one allele in particular increased substantially. The same allele was also found at a high frequency in an independently derived thiabendazole-selected field isolate. We present genetic evidence of selection at a P-glycoprotein locus during selection for benzimidzole resistance in H. contortus.

Successful Translation of a GMP-Based, Clinical Scale Selective Allodepletion Approach for Matched Donor-Recipient Pairs from Bench-to-Bedside.

Selective allodepletion (SD) is a strategy to eliminate host-reactive donor T-cells from hematopoietic stem cell allografts to prevent graft versus host disease (GvHD) while conserving useful donor immune functions. To overcome fluctuations in activation-based surface marker expression and achieve a more consistent and effective allodepletion, we investigated a photodepletion (PD) process targeting activation-based changes in p-glycoprotein that result in an altered efflux of the rhodamide-based photosensitizer TH9402. Expanded lymphocytes, generated using anti-CD3 and IL-2, were co-cultured with responder cells from HLA-matched or mismatched donors. Optimal results were achieved when co-cultured cells were incubated with 7.5 μM TH9402, followed by dye-extrusion and exposure to 5 Joule/cm2 light energy at 5x106 cells/ml. [Display omitted] In six mismatched stimulator-responder pairs, the median reduction of allo-reactivity was 474-fold (range, 43–864) when compared to the unmanipulated responder. Third-party responses were maintained with a median 1.4-fold (range, 0.9–3.3) reduction. In three matched pairs alloreactive helper T-lymphocyte precursors (HTLps) were reduced below 1:100,000 while third party responses remained above 1:10,000. CMV-specific T cells and responses to SEB were preserved. The intracellular TH9402 amount after extrusion correlated significantly with the depletion efficacy obtained against the original stimulator (r2=0.76; p=0.0235) but not against the 3rd party (r2=0.05; p=0.68). This establishes a clinical scale process capable of highly efficient, reproducible, selective removal of alloreactive lymphocytes from lymphocyte transplant products performed under current good manufacturing practice (cGMP) being reliably capable of generating at least 107/kg SD T cells for transfusion using a single PD session. In establishing this SD approach, we set out to design a technique that could ultimately be adopted in many cell transplant processing centers. Recent review of our Investigational New Drug (IND) application by the Food and Drug Administration (FDA) and review of our clinical protocol by our Institutional Review Board have been successful, and a clinical trial is in progress where recipients with hematological malignancies are given a myeloablative preparative regimen followed by a transplant of selected CD34+ cells together with 5 x 106 SD T cells/kg from HLA-matched sibling donors.

Impact of Curcumin-Induced Changes in P-Glycoprotein and CYP3A Expression on the Pharmacokinetics of Peroral Celiprolol and Midazolam in Rats

The aim of this study was to evaluate whether curcumin could modulate P-glycoprotein (P-gp) and CYP3A expression, and in turn modify the pharmacokinetic profiles of P-gp and CYP3A substrates in male Sprague-Dawley rats. Intragastric gavage of the rats with 60 mg/kg curcumin for 4 consecutive days led to a down-regulation of the intestinal P-gp level. There was a concomitant upregulation of hepatic P-gp level, but the renal P-gp level was unaffected. Curcumin also attenuated the CYP3A level in the small intestine but induced CYP3A expression in the liver and kidney. Regular curcumin consumption also caused the C(max) and area under the concentration-time curve (AUC(0-8) and total AUC) of peroral celiprolol (a P-gp substrate with negligible cytochrome P450 metabolism) at 30 mg/kg to increase, but the apparent oral clearance (CL(oral)) of the drug was reduced. Similarly, rats treated with curcumin for 4 consecutive days showed higher AUC (AUC(0-4) and total AUC) and lower CL(oral) for peroral midazolam (a CYP3A substrate that does not interact with the P-gp) at 20 mg/kg in comparison with vehicle-treated rats. In contrast, curcumin administered 30 min before the respective drug treatments did not significantly modify the pharmacokinetic parameters of the drugs. Analysis of the data suggests that the changes in the pharmacokinetic profiles of peroral celiprolol and midazolam in the rat model were contributed mainly by the curcumin-mediated down-regulation of intestinal P-gp and CYP3A protein levels, respectively.